University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA DEPARTMENT OF PLANT AND ENVIRONMENTAL BIOLOGY SURVEY OF THE MANGO TREE DECLINE DISEASE, CHARACTERISATION OF THE CAUSAL AGENT AND SOME ASPECTS OF THE BIOCONTROL OF THE PATHOGEN IN GHANA BY STEPHEN RODNEY COLEMAN (BSc. Hons. Agric., Cape Coast) (10508034) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL PLANT AND ENVIRONMENTAL BIOLOGY DEGREE JULY, 2016 0 University of Ghana http://ugspace.ug.edu.gh DECLARATION I hereby declare that except for reference to other peoples work which has been duly cited, this work is the result of my original research and that this thesis has neither in whole nor part been presented for another degree elsewhere. ……………………………………. …………………………………… Stephen Rodney Coleman Prof. G. T. Odamtten (Student) (Principal Supervisor) Date: ……………………………… Date: ……………………………… ………………………………….. ………………………………. Dr. Ebenezer Owusu Dr. J. O. Honger (Supervisor) (Supervisor) Date: ……………………………… Date: ……………………………… i University of Ghana http://ugspace.ug.edu.gh ABSTRACT Mango (Mangifera indica L.) production in Ghana has the potential of a being an economically viable venture capable of replacing cocoa as a major export earner. However, the crop is bedeviled by a miscellany of disease of the plant itself as well as the fruits. Recently a disease described as the ‘mango decline disease syndrome’ has been found in Ghana gradually taking a heavy toll of the crop in the field and a survey was carried out to substantiate this and prescribe a remedy. In this present study, a survey was carried out in five regions (Ashanti, Brong-Ahafo, Eastern, Greater- Accra, and Northern Regions) of Ghana with the view to documenting quantitatively the incidence and severity of the disease. It was also envisaged to authenticate and characterise the causal agent and to prescribe a biological control method in order to complement the current control methods using chemical sterilants. The survey was carried out with structured questionnaires using farmers as respondents. The percentage incident and severity index were determined by Standard Conventional method and the disease symptoms recorded in the field included: wilting of upper tips of the plants (tip dieback), shoot dieback, drying of the leaves and rolling of their margins, discolouration of vascular tissues, gummosis, cracking of bark, entire death of the plant on a 5 point hedonic scale. Cultural, morphological, physiological and colour characteristics were studied in vitro in Petri plates and using the conventional dry weight measurement technique. Molecular characterisation was done by polymerase chain reaction (PCR) and sequences and phylogenetic analysis of the ITS region. All the symptoms associated with mango decline disease (wilting of upper tips of the plants (tip dieback), shoot dieback, drying of the leaves and rolling of their margins, discolouration of vascular tissues, gummosis, cracking of bark, entire death of the plant) were observed on the diseased trees. The disease incidence on the local mango variety varied from 62.5% (Greater-Accra) to 70% in the Ashanti region but was severer (p≤0.05) (62.5-70.0%) on the local mango variety than the exotic Kent mango variety. The disease severity followed almost the same trend as the percentage incidence. The disease occurred in all the surveyed agro-ecological zones (Coastal savanna, Semi-deciduous forest, Transitional, and Guinea savanna zones) and was again higher on the local mango variety (60.0-74.0%) as compared to 13.33-20.0% in the exotic mango. The causative agent was recovered from 95% of the symptomatic mango plant tissue. Artificial inoculations confirmed the pathogenicity of the isolated pathogen on both local variety and Kent ii University of Ghana http://ugspace.ug.edu.gh (exotic) variety and induced similar disease symptoms which was severer (p≤0.05) on the local mango (Incidence 71.0%; Severity index 2.0) than the exotic Kent variety (Incidence 57.0%; Severity index 1.5). The fungi grew well on all the test media (natural and synthetic) and produced unique morphological, cultural and colour characteristics akin to the pathogen. Aqueous, ethyl acetate and petroleum ether extracts of local mango bark, local mango leaves, Kent (exotic) mango bark, Kent mango leaves, pine needles (Psuedotsuga spp.) and Egyptian date palm seeds (Phoenix sp.) supported vegetative growth of the pathogen to different extents but was best in the aqueous extract. Continuous light (75 lux intensity), continuous darkness, alternating 12 h light/12 h dark regimes produce nearly the same effect on the vegetative growth and colour of the culture which changed from white, to grey to black in 12-21 days. The fruit juice extract from local mango, exotic Kent mango fruit extract and soil extract also supported good vegetative growth of the pathogen showing that all the plant parts tested could support the survival of the pathogen. The mycelium was hyaline after 2 days and remained non-septate with a diameter of 2.0±1.3 µm and thereafter increased in diameter 2.0-15.0±1.5 µm. Septation occurred after 2 days. Rounding off of mycelium to form “chlamydospore-like” structures after 21 days was observed on some media but in the case of local mango bark, large “turbinate-like” cells with thickened walls were formed. The different light regimes employed did not influence the formation of these structures. The natural media, local mango fruit juice, Kent (exotic) mango fruit juice did not support conidia formation by the mycelium of the pathogen identified on the basis of cultural, morphological and physiological characteristics as Lasiodiplodia theobromae Griff and Manubl. But Soil extract agar, Potato Dextrose Agar and OGYE produced conidia of dimension 11.0-12.0±3.5 µm wide and 20.0-32.0±4.2 µm long which compared favourably with that reported in the literature for L. theobromae. PCR using universal primer pair (ITS1/ITS4) and sequence and phylogenetic analysis of the amplified ITS region showed all isolates from Ghana clustering together with the type strain of L. theobromae and other L. theobromae isolates which confirms that the pathogen from Ghana is L. theobromae. Aqueous extract of the leaf of Chromolaena odorata, Azadirachta indica and seeds of Carica papaya significantly depressed vegetative growth of L. theobromae at high concentration (undiluted-1:1 v/v) in the order of C. odorata = A. indica < Carica papaya. Practical implications of these findings are discussed and future studies suggested. iii University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is dedicated to the Glory of Almighty God and to all members of my family, especially in loving memory of my grandmother, Mrs. Agnes Yawson. iv University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS My first and foremost thanks go to the Almighty Father for His mercy, love and strength that sustained me throughout the period of my study. I would like to extend my sincerest appreciations to my supervisors, Prof. G. T. Odamtten, Dr. J. O. Honger and Dr. E. Owusu for their guidance and supervision as I carried out this research work. I am thankful for the assistance in diverse ways from Dr. M. Wiafe-Kwagyan, Mr. Baako, Mr. Akwetey, the Head of Department, Prof. Asante, and the entire Plant and Environmental Biology department staff. I am also grateful to the University of Ghana through the Office of Research and Innovation Development, ORID, for financial support for my research work through the project URF/8/ILG- 053/2014-2015. To many others whose names have not been mentioned but who contributed in diverse ways towards the completion of this research work, I say may God bless you all. v University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS Pages DECLARATION ................................................................................................................................ i ABSTRACT ....................................................................................................................................... ii DEDICATION .................................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................................... v TABLE OF CONTENTS .................................................................................................................. vi LIST OF FIGURES ......................................................................................................................... xii LIST OF MAPS .............................................................................................................................. xiii LIST OF TABLES .......................................................................................................................... xiv LIST OF PLATES ......................................................................................................................... xvii LIST OF ABBREVIATIONS .......................................................................................................... xx CHAPTER 1 ...................................................................................................................................... 1 INTRODUCTION .......................................................................................................................... 1 CHAPTER 2 ...................................................................................................................................... 6 LITERATURE REVIEW ............................................................................................................... 6 Mango: Origin, spread and cultivars .......................................................................................... 6 Phytochemicals in mango ......................................................................................................... 10 Mango production and its constraints ....................................................................................... 11 Mango tree decline disease: symptoms, predisposing factors, incidence, severity, and economic importance ................................................................................................................ 14 Aetiology of mango tree decline disease .................................................................................. 18 Identification of Lasiodiplodia theobromae ............................................................................. 20 Control of the mango tree decline syndrome............................................................................ 24 CHAPTER 3 .................................................................................................................................... 30 MATERIALS AND GENERAL METHODS ............................................................................. 30 Field survey of exotic and local mango variety farms .............................................................. 30 Incidence and severity of mango tree decline disease in Ghana in 2015 ................................. 34 Materials ................................................................................................................................... 34 Preparation of extracts .............................................................................................................. 35 Media composition and preparation ......................................................................................... 37 General methods ....................................................................................................................... 39 Isolation of the causal agent ..................................................................................................... 40 vi University of Ghana http://ugspace.ug.edu.gh Pathogenicity test ...................................................................................................................... 41 Characterisation of the isolated causal agent ............................................................................ 42 CHAPTER 4 .................................................................................................................................... 54 EXPERIMENTAL PROCEDURE .............................................................................................. 54 Field survey to assess the mango tree decline disease in the Administrative Regions of Ghana in 2015 ...................................................................................................................................... 54 Disease incidence and severity of the mango tree decline disease in the Administrative regions of Ghana in 2015 ...................................................................................................................... 55 Disease incidence and severity of the mango tree decline disease in the different agro- ecological zones of Ghana in 2015 ........................................................................................... 55 Cultural, morphological and physiological characteristics of the causative organism of the mango tree decline disease in Ghana ........................................................................................ 56 a. Radial growth of L. theobromae on different aqueous extracts of different plants parts of different origins incubated under alternating 12 h light and 12 h dark regimes at 28±1⁰C for 72 h……………………………………………………………………………………………….57 b. Radial growth of L. theobromae on ethyl acetate extracts of different plants parts of different origins incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 72 h……………………………………………………………………………………………….58 c. Radial growth of L. theobromae on petroleum ether extracts of different plants parts of different origins incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 72 h……………………………………………………………………………………………58 d. Radial growth of L. theobromae on fruit juice agar of local mango, Kent mango and soil extract agar under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days ................... 59 Vegetative growth and sporulation of L. theobromae in three different media (Czapek-Dox Broth, Oxytetracycline Glucose Yeast Extract Broth, Potato Dextrose Broth) at 28±1⁰C under different light and dark regimes for 4 days ............................................................................... 59 Vegetative growth and sporulation of L. theobromae in aqueous extract of leaves, bark and seeds of plants parts of different species incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days ................................................................................................... 60 Vegetative growth and sporulation of L. theobromae in ethyl acetate extract of leaves, bark and seeds of plants parts of different species incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days ................................................................................................... 60 Vegetative growth and sporulation of L. theobromae in petroleum ether extracts of leaves, bark and seeds of plants parts of different species incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days ........................................................................................... 61 Vegetative growth and sporulation of L. theobromae in fruit juices of Kent mango variety and local mango variety soil extract broths under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days ..................................................................................................................... 61 vii University of Ghana http://ugspace.ug.edu.gh Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media under continuous light (75 lux intensity) at 28±1⁰C for 21 days .......... 62 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media under continuous darkness at 28±1⁰C for 21 days ................................ 62 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media under alternating 12 h light/12 h darkness light regimes at 28±1⁰C for 21 days ...................................................................................................................................... 63 Colour change of the mycelium of L. theobromae growing on solid aqueous extract media of local mango bark, local mango leaves, Kent mango variety leaves, Kent mango variety bark, pine needles and Egyptian date palm seeds at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days ........................................................................................... 63 Colour change of the mycelium of L. theobromae growing on solid ethyl acetate extract media of local mango bark, local mango leaves, Kent mango variety leaves, Kent mango variety bark, pine needles and Egyptian date palm seeds at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days ........................................................................................... 64 Colour change of the mycelium of L. theobromae growing on solid mango fruit juice and soil extract agar at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days . 64 Colony morphology of L. theobromae on different media used in Experiments 4 to 22. ........ 65 Hyphal morphology of L. theobromae on different cultural formulations ............................... 65 Sporulation and conidial (spore) dimension of L. theobromae growing on differently formulated synthetic and natural media incubated at different light and dark regimes for 21 days ........................................................................................................................................... 66 Pathogenicity test of the causative pathogen on host tissue ..................................................... 67 Molecular characterization of the causative agent to confirm the morphology identity .......... 67 Preliminary studies on the biocontrol of L. theobromae using biotoxins from aqueous plant extracts ...................................................................................................................................... 68 CHAPTER 5 .................................................................................................................................... 70 RESULTS..................................................................................................................................... 70 Field survey to assess the mango tree decline disease in the administrative regions of Ghana in 2015 .......................................................................................................................................... 70 Disease incidence and severity of mango tree decline disease in the Administrative Regions in Ghana in 2015 ........................................................................................................................... 78 Disease incidence and severity index of the mango tree decline disease in the different agro- ecological zones of Ghana ........................................................................................................ 82 Cultural, morphological and physiological characteristics of the causative organism of the mango tree decline disease in Ghana ........................................................................................ 85 a. Radial growth of L. theobromae on different aqueous extracts of plants of different origins incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 72 h ...... 90 viii University of Ghana http://ugspace.ug.edu.gh b. Radial growth of L. theobromae on different ethyl acetate extracts of plants of different origins incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 72 h ...... 90 c. Radial growth of L. theobromae on different petroleum ether extracts of plants of different origins incubated under alternating 12 h light/12 h darkness at 28±1⁰C for 72 h ..... 91 d. Radial growth of L. theobromae on fruit juice agar prepared from local mango and Kent mango extract and soil extract agar incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 72 h ...................................................................................................... 91 Vegetative growth of L. theobromae in three different commercially prepared media under different light/dark regimes at 28±1⁰C for 4 days .................................................................... 96 Vegetative growth and sporulation of L. theobromae in aqueous extracts of leaves, bark and seeds of plant parts from different species incubated under 12 h light/12 h dark regimes at 28±1⁰C for 4 days ................................................................................................................... 101 Vegetative growth and sporulation of L. theobromae in ethyl acetate extracts of leaves, bark and seeds of plant parts from different species incubated under 12 h light/12 h darkness regimes at 28±1⁰C for 4 days ................................................................................................. 103 Vegetative growth and sporulation of L. theobromae in petroleum ether extracts of leaves, bark and seeds of plant parts from different species incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days ......................................................................................... 105 Vegetative growth of L. theobromae in fruit juices of Kent mango variety and local mango variety and soil extract broths under 12 h light/12 h darkness regimes at 28±1⁰C for 4 days 107 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media incubated under continuous light (75 lux intensity) at 28±1⁰C for 21 days ......................................................................................................................................... 109 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media incubated under continuous darkness at 28±1⁰C for 21 days .............. 112 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 21 days .............................................................................................................................. 115 Colour change of the mycelium of L. theobromae growing on solid aqueous extract media of local mango bark, local mango leaves, Kent mango variety leaves, Kent mango variety bark, pine needles and Egyptian date palm seeds at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days ......................................................................................... 118 Colour change of the mycelium of L. theobromae growing on solid ethyl acetate extract media of local mango bark, local mango leaves, Kent mango variety leaves, Kent mango variety bark, pine needles and Egyptian date palm seeds at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days ......................................................................................... 121 Colour change of the mycelium of L. theobromae growing on solid mango fruit juice and soil extract agar at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days124 ix University of Ghana http://ugspace.ug.edu.gh Colony morphology of L. theobromae on different mycological media used in these Experiments ............................................................................................................................ 126 Hyphal morphology of L. theobromae under different cultural formulations ........................ 128 Sporulation in culture and conidial dimension of L. theobromae growing on differently formulated synthetic and natural media incubated at different light/dark regimes at 28±1⁰C for 25 days .................................................................................................................................... 146 Pathogenicity test of the causative pathogen of mango tree decline syndrome on host plant 153 a. Molecular characterization of the causative agent (L. theobromae) of the mango tree decline disease in Ghana ......................................................................................................... 158 b. Sequences and phylogenetic analysis of the ITS region ................................................. 158 Preliminary studies on the biocontrol of L. theobromae using the biotoxins of the aqueous extracts of three plants ............................................................................................................ 162 CHAPTER 6 .................................................................................................................................. 173 DISCUSSION ............................................................................................................................ 173 CONCLUSION AND RECOMMENDATIONS ....................................................................... 186 SUMMARY ............................................................................................................................... 190 REFERENCES ........................................................................................................................... 194 APPENDICES ............................................................................................................................ 204 x University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Fig. 1. Key: Description of colony morphology used in the study………………………………..45 Fig. 2. Response (%) of farmers who have observed any of the typical symptoms of the mango tree decline disease on their farms in Ghana.………………………… ………………………….........76 Fig. 3. Response (%) of farmers on the possible cause of the mango tree decline disease on their farms in Ghana……………………………………………………………………………………..77 Fig. 4a. Selected hourly radial growth of L. theobromae on selected commercial synthetic media at 28±1⁰C under constant light (75 lux intensity)…………………………………………………….87 Fig. 4b. Radial growth of L. theobromae on selected commercial synthetic media at 28±1⁰C under constant light (75 lux intensity) for 72 h…………………………………………………………..87 Fig. 5a. Selected hourly radial growth of L. theobromae on selected commercial synthetic media at 28±1⁰C under constant darkness…………………………………………………………………..88 Fig. 5b. Radial growth rate of L. theobromae on selected commercial synthetic media at 28±1⁰C under constant darkness for 72 h…………………………………………………………………..88 Fig. 6a. Selected hourly radial growth of L. theobromae on selected commercial synthetic media at 28±1⁰C under alternating 12 h light and 12 h dark………………………………………………..89 Fig. 6b. Radial growth rate of L. theobromae on selected commercial synthetic media 28±1⁰C under alternating 12h light and darkness for 72 h…………….………………………………………….89 Fig. 7a. Selected hourly radial growth of L. theobromae on aqueous extracts of selected plant materials at 28±1⁰C under alternating 12 h light and 12 h dark…………………………………...92 Fig. 7b. Radial growth of L. theobromae on aqueous extracts of selected plant materials at 28±1⁰C under alternating 12 h light and darkness regimes for 72 h……………………………………….92 Fig. 8a. Radial growth of L. theobromae on ethyl acetate extracts of selected plant materials at 28±1⁰C under alternating 12h light and darkness…………………………………………………93 Fig. 8b. Radial growth rate of L. theobromae on ethyl acetate extracts of selected plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 72 h…………………………….…….93 Fig. 9a. Selected hourly radial growth of L. theobromae on petroleum ether extracts of selected plant materials at 28±1⁰C under alternating 12 h light and 12 darkness…………………………….….94 xi University of Ghana http://ugspace.ug.edu.gh Fig. 9b. Radial growth rate of L. theobromae on petroleum ether extracts of selected plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness regimes for 72 h……………………….94 Fig. 10a. Selected hourly radial growth of L. theobromae on mango fruit juice and soil extracts at 28±1⁰C under alternating 12 h light and 12 h darkness…………………………………………...95 Fig. 10b. Radial growth of L. theobromae on mango fruit juice and soil extracts at 28±1⁰C under alternating12 h light and 12 h darkness for 72 h………………………………………………….95 Fig. 11. Neighbour joining tree drawn with the nucleotide sequences of the ITS region of 8 different isolates of Lasiodiplodia in Ghana……………………………………………………………….160 Fig. 12a. Influence of PDA amended with the indicated varying concentrations of aqueous extract Chromolaena odorata leaves on radial growth of L. theobromae……………………………………166 Fig. 12b. Influence of PDA amended with varying concentrations of aqueous extract of Chromolaena odorata leaves on the radial growth of L. theobromae at 28±1⁰C………………..166 Fig. 13a. Influence of PDA amended with varying concentrations of aqueous extract of Azadirachta indica leaves on the radial growth of L. theobromae at 28±1⁰C…………………………………167 Fig. 13b. Influence of PDA amended with varying concentrations of aqueous extract of Azadirachta indica leaves on the radial growth of L. theobromae at 28±1⁰C……………………………….…167 Fig. 14a. Influence of PDA amended with varying concentrations of aqueous extract of Carica papaya seeds on the radial growth of L. theobromae at 28±1⁰C…………………………………168 Fig. 14b. Influence of PDA amended with the indicated varying concentrations of aqueous extract of Carica papaya seeds on the radial growth of L. theobromae at 28±1⁰C……………………...168 Fig. 15. Influence of PDA amended with the indicated varying concentrations of aqueous extract Chromolaena odorata leaves on the vegetative growth of L. theobromae at 28±1⁰C for 5 days..170 Fig. 16. Influence of PDA amended with varying concentrations of aqueous extract Azadirachta indica leaves on the vegetative growth of L. theobromae at 28±1⁰C for 5 days………………...171 Fig. 17. Influence of PDA amended with varying concentrations of aqueous extract of Carica papaya seeds on the vegetative growth of L. theobromae at 28±1⁰C for 5 days……………………..…..172 xii University of Ghana http://ugspace.ug.edu.gh LIST OF MAPS Map 1. A map of Ghana showing the disease incidence and severity in selected five administrative regions of Ghana in 2015………………………………………………………………………….80 Map 2. A map of Ghana showing the disease incidence and severity in four agro-ecological zones of Ghana in 2015…………………………………………………………………………………..84 xiii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 1. Disease severity rating scale used for the assessment of disease severity in different mango farms in Ghana. ……………………………………………………………………………………33 Table 2. Mean percentage disease incidence and severity index of mango tree decline disease in 36 mango farms in five Administrative regions in Ghana in 2015. …………………………………..81 Table 3. Mean percentage incidence and severity index of mango tree decline disease in 36 mango farms in four agro-ecological zones of Ghana in 2015. …………………………………………..83 Table 4. Vegetative growth and sporulation of L. theobromae in the indicated liquid media at 28±1⁰C under continuous light for 4 days.………… ……………………………………………………...98 Table 5. Vegetative growth and sporulation of L. theobromae in the indicated liquid media at 28±1⁰C under continuous darkness for 4 days.………………………………………………………….…99 Table 6. Vegetative growth and sporulation of L. theobromae the indicated liquid media at 28±1⁰C under alternating 12 h light and 12 h darkness for 4 days. ………………………………………100 Table 7. Vegetative growth and sporulation of L. theobromae in aqueous extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and darkness. …………………………………102 Table 8. Vegetative growth and sporulation of L. theobromae in ethyl acetate extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness. …………….………..104 Table 9. Vegetative growth and sporulation of L. theobromae in petroleum ether extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness.………………………106 Table 10. Vegetative growth and sporulation of L. theobromae in mango fruit juice and soil extracts media at 28±1⁰C under 12 h alternating light and 12 h darkness regimes.………………….…...108 Table 11. Morphology of the colonies of L. theobromae culture on indicated synthetic media at 28±1⁰C under three different light regimes. ………………….………………….………………127 xiv University of Ghana http://ugspace.ug.edu.gh Table 12. Sporulation of L. theobromae on the indicated commercial synthetic media at 28±1⁰C under continuous darkness for 25 days. ………………….…………………..…………………150 Table 13. Sporulation of L. theobromae on selected commercial synthetic media at 28±1⁰C under alternating 12 h light and 12 h darkness for 25 days. ………………….…………….………….151 Table 14. Sporulation of L. theobromae on mango fruit juice and soil extracts solid media at 28±1⁰C under alternating 12 h light and 12 h darkness for 25 days. ………………………………….…152 Table 15. Percentage incidence and severity of the pathogenicity test of mango tree decline disease on mango plants in a screen house………………….………………….…………………………157 Table 16. ITS1 grouping of L. theobromae isolates used in the study collected from different locations in Ghana.………………….……………………………161 xv University of Ghana http://ugspace.ug.edu.gh LIST OF PLATES Plate 1. Kent mango variety tree in the Northern Region of Ghana………………………………..7 Plate 2. Typical symptoms of the mango tree decline disease observed in the field in Ghana……72 Plate 3. Typical symptoms of the mango tree decline disease observed in the field in Ghana……73 Plate 4. Typical symptoms of the mango tree decline disease observed in the field in Ghana……74 Plate 5. Colour change of colony of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under continuous light…………………………………………………………………………………..110 Plate 6. Colour change of colony of L. theobromae on Dichloran Rose-Bengal Chloramphenicol Agar (DRBC) at 28±1⁰C under continuous light…………………………………………………110 Plate 7. Colour change of colony of L. theobromae on oxytetracycline glucose yeast agar (OGYE) at 28±1⁰C under continuous light………………………………………………………………...111 Plate 8. Colour change of colony of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under continuous light……………………………………………………………………………….….111 Plate 9. Colour change of colony of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under continuous darkness………………………………………………………………………………113 Plate 10. Colour change of colony of L. theobromae on Dichloran Rose-Bengal Chloramphenicol Agar (DRBC) at 28±1⁰C under continuous darkness…………………………………………….113 Plate 11. Colour change of colony of L. theobromae on Oxytetracycline Glucose Yeast Agar (OGYE) at 28±1⁰C under continuous darkness…………………………………………………..114 Plate 12. Colour change of colony of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under continuous darkness………………………………………………………………………..114 Plate 13. Colour change of colony of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under alternating 12 h light and 12 h darkness………………………………………………………….116 Plate 14. Colour change of colony of L. theobromae on Dichloran Rose-Bengal Chloramphenicol Agar (DRBC) at 28±1⁰C under alternating 12 h light and 12 h darkness………………………..116 Plate 15. Colour change of colony of L. theobromae on Oxytetracycline Glucose Yeast Agar (OGYE) at 28±1⁰C under alternating 12 h light and 12 h darkness……………………………..117 Plate 16. Colour change of colony of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under 12 h alternating light and 12 h darkness…………………………………………………..117 Plate 17. Colour change of L. theobromae on aqueous extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 5 days…………………………………….…119 xvi University of Ghana http://ugspace.ug.edu.gh Plate 18. Colour change of colony of L. theobromae on aqueous extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 darkness for 14 days……………………………..120 Plate 19. Colour change of colony of L. theobromae on ethyl acetate extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 5 days………………….122 Plate 20. Colour change of colony of L. theobromae on ethyl acetate extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 14 days………………...123 Plate 21. Colour change of colony of L. theobromae on indicated natural media at 28±1⁰C under alternating 12 h light and 12 h darkness for 5 days………………………………………………125 Plate 22. Colour change of colony of L. theobromae on indicated natural media at 28±1⁰C under alternating 12 h light and 12 h darkness for 14 days……………………………………………..125 Plate 23. Photograph showing the hyphae of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under continuous light for 21 days……………………………………………………...131 Plate 24. Photograph showing the hyphae of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under continuous light for 21 days…………………………………………………………..…..131 Plate 25. Photograph showing the hyphae of L. theobromae on Dichloran Rose-Bengal Chloramphenicol agar (DRBC) at 28±1⁰C under continuous light for 21 days…………………132 Plate 26. Photograph showing the hyphae of L. theobromae on Oxytetracycline Glucose Yeast Extract agar (OGYE) at 28±1⁰C under continuous light for 21 days……………………………132 Plate 27. Photograph showing the hyphae of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under continuous darkness for 21 days………………………………………………….133 Plate 28. Photograph showing the hyphae of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under continuous darkness for 21 days…………………………………………………….…….133 Plate 29. Photograph showing the hyphae of L. theobromae on Dichloran Rose-Bengal Chloramphenicol Agar (DRBC) at 28±1⁰C under continuous darkness for 21 days…………….134 Plate 30. Photograph showing the hyphae of L. theobromae on Oxytetracycline Glucose Yeast Extract Agar (OGYE) at 28±1⁰C under continuous darkness for 21 days……………………….134 Plate 31. Photograph showing the hyphae of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under alternating 12h light and 12 h darkness…………………………………………..135 Plate 32. Photograph showing the hyphae of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under alternating 12 h light and 12 h darkness…………………………………………………..135 Plate 33. Photograph showing the hyphae of L. theobromae on Dichloran Rose-Bengal Extract agar (DRBC) at 28±1⁰C under alternating 12 h light and 12 h darkness……………………………..136 xvii University of Ghana http://ugspace.ug.edu.gh Plate 34. Photograph showing the hyphae of L. theobromae on Oxytetracycline Glucose Yeast Extract Agar (OGYE) at 28±1⁰C under alternating 12 h light and 12 h darkness………………136 Plate 35. Photograph showing the hyphae of L. theobromae on aqueous extract of local mango bark (ALB) at 28±1⁰C under alternating 12 h light and 12 h darkness………………………………..137 Plate 36. Photograph showing the hyphae of L. theobromae on aqueous extract of local mango leaves (ALL) at 28±1⁰C under alternating 12 h light and 12 h darkness………………………………...137 Plate 37. Photograph showing the hyphae of L. theobromae on aqueous extract of Kent mango bark (AKB) at 28±1⁰C under alternating 12 h light and 12 h darkness……………………………….138 Plate 38. Photograph showing the hyphae of L. theobromae on aqueous extract Kent mango leaves (AKL) at 28±1⁰C under alternating 12 h light and 12 h darkness…………………………….....138 Plate 39. Photograph showing the hyphae of L. theobromae on aqueous extract of pine needles (APN) at 28±1⁰C under alternating 12 h light and 12 h darkness……………………………….139 Plate 40. Photograph showing the hyphae of L. theobromae on aqueous extract of Egyptian date palm seeds (ADS) at 28±1⁰C under alternating 12 h light and 12 h darkness…………………...139 Plate 41. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of local mango bark (ELB) at 28±1⁰C under alternating 12 h light and 12 h darkness…………………………..140 Plate 42. Photograph showing the hyphae of L. theobromae on ethyl acetate extracts of local mango leaves (ELL) at 28±1⁰C under alternating 12 h light and 12 h darkness…………………………140 Plate 43. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of Kent bark (EKB) at 28±1⁰C under alternating 12 h light and 12 h darkness………………………………..141 Plate 44. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of Kent leaves (EKL) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days……………….…..141 Plate 45. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of pine needles (EPN) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days……………………142 Plate 46. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of Egyptian date palm seeds (EDS) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days ………142 Plate 47. Photograph showing the hyphae of L. theobromae on petroleum ether extract of local mango bark (PLB) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days………143 Plate 48. Photograph showing the hyphae of L. theobromae on petroleum ether extract of local mango leaves (PKB) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days…….143 Plate 49. Photograph showing the hyphae of L. theobromae on petroleum ether extract of Kent bark (PKB) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days…………………...144 xviii University of Ghana http://ugspace.ug.edu.gh Plate 50. Photograph showing the hyphae of L. theobromae on petroleum ether extract of Kent leaves (PKB) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days……………144 Plate 51. Photograph showing the hyphae of L. theobromae on petroleum ether extract of pine needles (PPN) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days…………..145 Plate 52. Photograph showing the hyphae of L. theobromae on petroleum ether extract of Egyptian date palm seeds (PDS) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days….145 Plate 53. Sporulation of L. theobromae on Potato Dextrose Agar (PDA) media at 28±1⁰C under continuous darkness for 25 days………………………………………………………………….148 Plate 54. Sporulation of L. theobromae on DRBC and PDA solid media at 28±1⁰C under alternating 12 h light and 12 h darkness for 25 days……………………………………………..…………..148 Plate 55. Sporulation of L. theobromae on Soil Extract Agar at 28±1⁰C under alternating 12 h light and 12 h darkness for 25 days…………………………………………………………………….149 Plate 56. A close-up photographs showing the typical symptoms of the mango tree decline disease observed on L. theobromae-inoculated plants in the screened house after 40 days…………154-155 Plate 57. Photograph showing symptoms of the mango tree decline disease observed on mango plants in the screen house for 40 days……………………………………………………………156 Plate 58. A gel showing the approximately 347 bp product amplified from Lasiodiplodia species isolated from mango in Ghana……………………………………………………………………159 Plate 59. Influence of varying concentrations of the aqueous extracts of Chromolaena odorata leaves on the radial growth of L. theobromae at 28±1⁰C for 3 days…………………………..………..164 Plate 60. Influence of varying concentrations of the aqueous extracts of Azadirachta indica leaves on the radial growth of L. theobromae at 28±1⁰C for 3 days…………………………………….164 Plate 61. Influence of varying concentrations of the aqueous extracts of Carica papaya seeds on the radial growth of L. theobromae at 28±1⁰C for 3 days…………………………………………….165 xix University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS PDA - Potato Dextrose Agar CDA - Czapek-Dox Agar DRBC - Dichloran Rose-Bengal Chloramphenicol agar OGYE - Oxytetracycline Glucose Yeast Extract Agar WA - Water Agar PDB - Potato Dextrose Broth CDB - Czapek- Dox Broth OGYE-B - Oxytetracycline Glucose Yeast Extract Broth ALB - Aqueous extract from Local mango variety Bark ALL - Aqueous extract from Local mango variety Leaves AKB - Aqueous extract from Kent mango variety Bark AKL - Aqueous extract from Kent mango variety Leaves APN - Aqueous extract from Pine Needles ADS - Aqueous extract from Egyptian Date palm Seeds SEA - Soil Extract Agar ELB - Ethyl acetate extract from Local mango variety Bark ELL - Ethyl acetate extract from Local mango variety Leaves EKB - Ethyl acetate extract from Kent mango variety Bark EKL - Ethyl acetate from Kent mango variety Leaves EPN - Ethyl acetate extract from Pine Needles EDS - Ethyl acetate extract from Egyptian Date palm Seeds PLB - Petroleum ether extract from Local mango variety Bark PLL - Petroleum ether extract from Local mango variety Leaves PKB - Petroleum ether extract from Kent mango variety Bark PKL - Petroleum ether extract from Kent mango variety Leaves PPN - Petroleum ether extract from Pine Needles PDS - Petroleum ether extract from Egyptian Date palm Seeds JLF - Juice from Local mango variety Fruits JKF - Juice from Kent exotic mango variety Fruits xx University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 INTRODUCTION Mango (Mangifera indica L.) is a member of the flowering plant family, Anacardiaceae, in the order Sapindales. Related species include Bindjai (Mangifera caesia), Horse Mango (Mangifera foetida) and Kuweni mango (Mangifera odorata) (Susser and Schneider, 2001). Mango is found in the wild in India and Pakistan, where it is indigenous and several cultivated varieties have been introduced to other tropical regions of the world. It is one of the largest fruit-trees in the world, capable of growing to a height of 30 m and an average circumference of 3.6 to 4.2 m; sometimes reaching 6 m. Mango trees are long-lived, as some cultivars still fruit after 300 years. Notable among the cultivars are Kent, Keith, Palmer, Haden, Tommy Atkins and Irwin (Jedele et al., 2003; Susser and Schneider, 2001). The origin of the crop has been debated. It is largely believed that the crop originated in the Indo- Burma region and has been in cultivation for over 4000 years in the Southeastern Asia countries including Phillipines, Java, Indonesia, Thailand, Burma (now Myanmar), Malaysia and Sri Lanka. The crop was later introduced into East and West Africa, then to Brazil in the sixteenth century. It was introduced into Mexico in the nineteenth century and Florida in 1833 (Litz, 1998). Mangifera indica L. is one of the most important fruit crops in the tropics, and approximately makes up 50% of tropical fruits produced in the world (Jedele et al., 2003). Mango is the apple (or peach) of the tropics, and one of the most commonly eaten fruits in tropical countries around the world (Susser and Schneider, 2001). 1 University of Ghana http://ugspace.ug.edu.gh Worldwide production from mango orchards stood at 26,786,757 metric tonnes in 2008 with Pakistan producing 5%, India 50%, China 15%, Indonesia 7%, Brazil 4%, China 5%, Mexico 9% and Thailand 5% (FAO, 2008). The world’s largest producer of the crop is India followed by China and Thailand. Until 2005, Mexico was the leading exporter of mango worldwide. Total exports from Mexico amounted to 232,643 metric tonnes as compared to 286,775 metric tonnes from India in the 2009. The largest producer of the crop in Africa is Nigeria followed by Egypt; and Ghana producing modest amounts of the crop (FAOSTAT, 2009). Mango is a leading crop in most of the international markets and hence an important source of foreign exchange for most developing countries including Ghana. Currently in Ghana, the commercial production of mango which was previously concentrated in the coastal savanna zone is spreading to the other agro-ecological zones. Ghana has been exporting the crop for the past 20 years but is still not recognized as an important exporting country in international markets (Anon., 2004). Mango yield per acre is better than cocoa, while its cost of production per revenue generated on investment is better than cocoa (GNA, 2013). It is believed that if the major problems related to the production of the crop are properly handled, the mango crop has a potential of being the number one export earner of Ghana, thereby replacing cocoa (GNA, 2013). There are many adverse factors affecting the production of quality mango fruits in Ghana. These range from unfavourable weather conditions to incidence of pests and diseases. 2 University of Ghana http://ugspace.ug.edu.gh These diseases cause both qualitative and quantitative losses in the crop and bring added costs in the form of institution of control measures to the farmers (Arauz, 2000). Access to capital and improved planting materials of export varieties is also a challenging factor for many farmers in Ghana (Nimoh, 2007). Recently, another major factor impacting vitality and yield of mango is the mango tree decline syndrome, recognized in virtually all mango-producing regions of the world (Ramos et al., 1991; Anwar et al., 2012). Although fungi are the implicated inducers in many locations, abiotic stresses including host nutritional deficiencies are thought to play a role (Kazmi et al., 2005). It is known that these deficiencies predispose trees to infection by fungal pathogens including Botryodiplodia ribis and Physalospora sp. Nematodes have also been associated with presence of mango decline (Siddiqui, 2007). On the basis of the current research work done, it was concluded that the mango decline is a complex phenomenon of both biotic and abiotic factors (Sauer, 1981; McSorley et al., 1982; McSorley and Parrado, 1982; Schaffer et al., 1988; Ploetz et al., 1996; Jiskani, 2002). This syndrome has been associated with symptomatic tissues exhibiting bud necrosis, tip dieback, gummosis and vascular discolouration (Narasimhudu and Reddy, 1992; Sharma, 1993; Ploetz et al., 1996; Jiskani, 2002; Fateh et al., 2006), terminal and marginal necrosis of leaves, which ultimately lead to the death of the leaf blade (Al-Adawi et al., 2006; Saeed and Masood, 2008). The expression of symptoms usually starts with leaves necrosis, tip dieback, vascular discolouration, gummosis, branch callus formation, splitting or cracking of bark and death of plant. The above-mentioned 3 University of Ghana http://ugspace.ug.edu.gh symptoms may be found alone or in combination of two or more on the same plant (Ploetz et al., 1996; Iqbal et al., 2007). Acquisition of knowledge on the nature of the disease cannot be overemphasized. In designing sustainable management practices to increase productivity and fruit quality; raise incomes of farmers; alleviate poverty; meet domestic and international market standards, the approach should be environmentally friendly. However, very limited research has been done on this phenomenon in Ghana, although the disease is taking a heavy toll on the crop. Thus, there is the need for the nature and spread of the disease in the country to be elucidated and the causal agent identified and authenticated. Objectives of this thesis were to determine the nature of disease’s symptoms; its incidence and severity; isolate the causal pathogenic agent; characterize the causal agent(s); and to assess biotoxins from local plants in controlling the growth of the pathogen. 4 University of Ghana http://ugspace.ug.edu.gh Specific objectives of this study were to determine: 1. The nature of symptoms of the syndrome in Ghana by relying collectively on field survey, questionnaires and pathogenicity test. 2. The incidence and severity of the mango tree decline syndrome in Ghana by the use of disease score sheets during the survey. 3. The causal pathogenic agent. 4. The taxonomic characteristics of isolated causal agent. 5. The possible biocontrol of the isolated pathogen using biotoxins of known plant extracts. 5 University of Ghana http://ugspace.ug.edu.gh CHAPTER 2 LITERATURE REVIEW Mango: Origin, spread and cultivars Mango is a juicy stone fruit (drupe) belonging to the flowering plant family Anacardiaceae and genus Mangifera, consisting of numerous tropical fruiting trees, cultivated mostly for their edible fruits. The genus Mangifera belongs to the order Sapindales with 73 genera (c. 850 species), with a few representatives in temperate regions. The other distant relatives of Mangifera include cashew (Anacardium occidentale), gandaria (Bouea gandaria), pistachio (Pistacia vera), marula (Sclerocarya birrea), ambarella (Spondias cytherea), yellow mombin (Spondias mombin), red mombin (Spondias purpurea), imbu (Spondias tuberosa), dragon plums (Dracontomelum spp.) and kaffir plum (Harpepbyllum caffrum) (Maxwell et al., 1984; Samson, 1986). Mango (Mangifera indica L.) trees may reach 40 m or more in height and live for several hundred years (Plate 1). They bear rosettes of evergreen leaves (red or yellow at first) and dense panicles up to 30 cm long of small (5-10 mm) reddish or yellowish flowers. The inflorescence is a much- branched panicle bearing many very small (4 mm) greenish white or pinkish flowers. Both male and bisexual flowers are borne on the same tree. The flowers are radially symmetrical, and usually have 5 petals, streaked with red. There is usually only 1 fertile stamen per flower; the 4 other stamens are sterile. The flower has a conspicuous 5-lobed disc between the petals and stamens. The fruits, which range from 2.5 cm to more than 30 cm in length, depending on the cultivar, vary in shape (from round to oval, egg-shaped, or kidney-shaped) and color (green, yellow, red, or purple) with a dotted skin (Vaughan and Geissler, 1997; Mukherjee and Litz, 2009). 6 University of Ghana http://ugspace.ug.edu.gh 0.2 m Plate 1. Kent mango variety tree in the Northern Region of Ghana. 7 University of Ghana http://ugspace.ug.edu.gh Origin of mango There is considerable debate about the origin of mango, and in some cases, it has been a matter of speculation. Common mango (Mangifera indica L.) originated as alloploid and its native home was suggested as Eastern India, Assam to Burma or possibly further in the Malay region (Popenoe, 1920). Vavilov (1926) also suggested Indo-Burma region as the centre of origin. Introduction of superior types into Malay region from India is also an evidence of its origin in India. Based on detailed study of the history, phyto-geographical distribution of allied species, fossil records, and evidence of numerous wild and cultivated varieties in India, Mukherjee (1951) considered origin of genus Mangifera probably in Burma, Siam, Indo-China and the Malay peninsula, but the birth of common mango is in Assam-Burma region and not in Malay. According to De Candolle (1884), 'It is impossible to doubt that mango is a native of south Asia or of the Malay archipelago, when one comes across the multitude of varieties cultivated in those countries, the number of ancient names, in particular a Sanskrit name, its abundance in the gardens of Bengal, of Deccan peninsula, and of Ceylon (now Sri Lanka). Spread of mango Mango has spread almost over all the tropical areas towards the South and Southeast of Asia, Australia, Madagascar, East of Africa, Brazil and Central America. It is also grown in subtropical areas of favourable climate like Florida, South Africa, Israel, Cyprus and Egypt. As regard to the subtropics, this crop was probably introduced in the south of Africa in the 16 th century B.C., but it did not arrive at the Canary Islands and Madeira until the second half of the 18 th century and at the U.S.A. (Florida and Hawaii), Australia and Israel until the 19 th century. This fruit arrived at America by means of the Portuguese, who introduced it in Brazil in the 18 th 8 University of Ghana http://ugspace.ug.edu.gh century. The Portuguese also introduced it in West Africa. The Spaniards, on the other hand, contributed in the expansion of mangoes all over America, because they transported small producing trees from the Philippines to Mexico. The introduction of mango in Southern Spain might have not taken place until the 20 th century. On the other hand, the obtaining in 1910 in Florida of the excellent cultivar "Haden" marked the beginning of the modern development of this crop (FAOSTAT, 2000). Mango cultivars There are many hundreds of named mango cultivars, more than 500 varieties in India alone (Allen, 2006). Cultivars that are cultivated worldwide include Aloha, Brooks, Cambodiana, Carrie, Cooper, Doubikin, Gouveia, Haden, Julie, Kensington Pride, MacPherson, Manila, Sensation, Thomson, Winters and Zill. Popular cultivars grown in Ghana include Haden, Irwin, Julie, Keitt, Kent, Palmer and Tommy Atkins (Honger et al., 2014). The tree canopy of Haden cultivar spreads wide; fruits are large (700-800 g), regular ovate, yellow and almost covered with red; mild flavour with little fiber; and susceptible to anthracnose disease. Irwin mango trees are generally very small sized; medium sized fruits (350-400 g), elongated, ovate regular in form, orange yellow with deep blush, and fiberless. Slow growing Julie mango variety originated from the Trinidad. Its fruits are comparatively small (168-280 g), flat oblong, obliquely almost two-nosed, orange coloured, fibrous, juicy and sweet tasted. Keitt fruits are ovate with slightly oblique apex, green, flesh rich, fiber found only around seed, and strongly resists mildew disease; fruit production is relatively heavy and consistent. Kent, on the other hand has upright tree canopy; fruits are often large (~1.2 kg), regular ovate, greenish yellow with red shoulder, rich in flesh, and fiberless. Timmy Atkins originated from a seed planted in the 1920s at Fort Lauderdale, 9 University of Ghana http://ugspace.ug.edu.gh Florida. It has a dense tree canopy. Fruits are medium to large, ~450 g with thick skin, regular ovate, orange-yellow covered with red and heavy purple bloom; juicy with moderate fiber content, and fair to good quality. Fruits of Tommy Atkins are often valued for their very long shelf life and tolerance of handling and transportation with little or no bruising or degradation (Susser and Schneider, 2001). Many desired cultivars are monoembryonic and must be propagated by grafting or they do not breed true. Cultivars that excel in one climate may fail elsewhere. For example, Indian cultivars such as Julie, a prolific cultivar in Jamaica, require annual fungicide treatments to escape the lethal fungal disease anthracnose in Florida (Susser and Schneider, 2001). The current world market is dominated by the cultivar Tommy Atkins, a seedling of Haden that first fruited in 1940 in southern Florida and was initially rejected commercially by Florida researchers (Susser and Schneider, 2001). Growers and importers worldwide have embraced the cultivar for its excellent productivity and disease resistance, long shelf life, transportability, size, and appealing color (Mintz, 2008). Phytochemicals in mango Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties. Phytochemical and nutrient content appear to vary across mango cultivars. Rocha et al. (2007) isolated up to 25 different carotenoids from mango pulp, the densest of which was beta- carotene, which accounts for the yellow-orange pigmentation of most mango cultivars. Some of the phytochemicals that are present in mango peel and pulp, such as the triterpene lupeol is under basic research for its potential biological effects. Mango leaves have significant polyphenol content, including xanthonoids, mangiferin and gallic acid (Barreto et al., 2008). An extract of mango branch bark also contains many polyphenols, mangiferine and 16% to 20% tannin (USDA NRCS, 2016). 10 University of Ghana http://ugspace.ug.edu.gh Mango contains phytochemicals such as: 2-octene, alanine, alpha-phellandrene, alpha-pinene, ambolic-acid, ambonic-acid, arginine, ascorbic-acid, beta-carotene beta-pinene, carotenoids, furfurol, gaba, gallic-acid, gallotannic-acid, geraniol, histidine, isoleucine, isomangiferolic-acid, kaempferol, limonene, linoleic-acid, mangiferic-acid, mangiferine, mangiferol, mangiferolic-acid, myristic-acid, neo-beta-carotene-b, neo-beta-carotene-u, neoxanthophyll, nerol, neryl-acetate, oleic- acid, oxalic-acid, p-coumaric-acid, palmitic-acid, palmitoleic-acid, pantothenic-acid, peroxidase, phenylalanine, phytin, proline, quercetin, and xanthophyll mangiferin (a pharmacologically active flavonoid, a natural xanthone C-glycoside) and are extracted from mango at high concentrations from young leaves (172 g/kg), bark (107 g/kg), and from old leaves (94 g/kg). (Shah et al., 2010). Mangiferin has a number of pharmacological actions and possible health benefits, which include antifungal, anti-microbal, anti-inflamatory and anti-viral. In ripe mangoes, volatile compounds (40 to 70 ppm) are ocimene, limonene, α-terpineol, 3-carene, β-selinene and myrcene. The yellow colour of ripe mangoes is due to about 30 ppm β-carotene. The bark and twigs possess 16% to 20% tannin, alkaloids, phenol, and flavonoids (Shah et al., 2010). Mango production and its constraints Mango plant is one of the most important fruit crops in the tropics, and approximately makes up 50% of tropical fruits produced in the world (Jedele et al., 2003). Mango is currently produced in most sub-tropical and tropical regions of the world and until recently, it was an exotic specialty crop in most markets in the United States and Europe (Galinsky and Law, 1998). A single mature mango tree can produce 2000 to 2500 ripe fruits (Jiron and Headström, 1985). Mango performs best at elevations from 0-1200 m, with a pronounced rainy season for vegetative growth, a dry season for flowering and fruiting, and on well-drained soils ranging in pH between 5.5 11 University of Ghana http://ugspace.ug.edu.gh to 7.5. Individual trees often flower irregularly; some trees do not flower for periods of 10-20 years, sometimes even longer, others less than 10 years old may flower and fruit regularly every year. Thereafter, most mangoes tend toward alternate, or biennial, bearing. Branches that fruit one year may rest the next, while branches on the other side of the tree will bear. Blooming is strongly affected by weather, dryness stimulating flowering and rainy weather discouraging it (Litz, 1998). The world mango production area was about 3,500,753 ha in 2008 with 61% located in India and 5% in Pakistan (FAO, 2008). In 2008, Pakistan orchards had climbed to 166,223 ha over that of 88,272 ha in 1995 but the production stood at the same level (FAO, 1995 and 2008). India is the world largest producer of mango producing about 70% of total world production. Other important mango producing countries include Thailand, China, Mexico and Brazil. Until 2005, Mexico used to be the leading exporter of mango worldwide, but overtaken recently by India. Total exports from Mexico amounted to 232,643 metric tonnes as compared to 286,775 metric tonnes from India in the 2009. Other important exporting countries in the world include the Philippines, Thailand and Ecuador (FAOSTAT, 2009). Currently in Africa, Kenya is the largest producer with 553,710 metric tonnes, followed by Egypt with 505,741 metric tonnes in 2010. Nigeria used to be the largest producer of the crop with about 730,000 metric tonnes in 2005. West African countries such as Cote d’Ivoire and Ghana produce modest quantities of this fruit crop (FAOSTAT, 2009). The production, trade and consumption of mango fruits have increased significantly both domestically and internationally due to its attractive sensory properties and growing recognition of their nutritional and therapeutic properties (Bicas et al., 2011). It is nutritionally rich in 12 University of Ghana http://ugspace.ug.edu.gh carbohydrates, vitamins A and C, amino acids, fatty acids and proteins (Saleem and Akhtar, 1989). Mango is the apple (or peach) of the tropics, and one of the most commonly eaten fruits in many tropical countries (Susser and Schneider, 2001). This unique fruit is a leading crop in most of the international markets and hence is an important source of foreign exchange for most developing countries including Ghana. Currently in Ghana, the commercial production of mango which was previously concentrated in the coastal savanna zone is spreading to the other agro-ecological zones. Diseases affecting the production of mango worldwide include powdery mildew (Oidium mangiferae), fruit rot (Aspergillus niger), anthracnose (Colletotrichum gloeosporioides), stem blight or dieback (Ceratocystis fimbriata and Lasiodiplodia theobromae), root rot (Rhizoctinia and Fusarium species) and tip dieback (Fusarium equiseta, Alternaria alternata, Aspergillus niger and Rhizopus nigricans) are recorded as fungal diseases; bacterial black spot (Xanthomonas campestris pv. Mangiferae indica); and malformation of mango inflorescence are reported (Khalid et al., 2002). Among all these diseases infecting mango, a disease complex known as ‘mango sudden death (decline) syndrome/mango quick decline/mango decline disease’ is the most recent severe threat to the mango industry (Saeed and Masood, 2008). This syndrome is adversely affecting the vitality and yield of mango and it is recognized in virtually all mango-producing regions of the world (Ramos et al., 1991; Anwar et al., 2012) including Ghana. 13 University of Ghana http://ugspace.ug.edu.gh Mango tree decline disease: symptoms, predisposing factors, incidence, severity and economic importance Symptoms and signs of the mango tree decline disease Infected leaf turns brown and its margins roll upwards. Leaves scorch and fall, leaving a dead branch. Twigs die from the tips back into old wood, giving a scorched appearance to the limb. The young green twigs start withering first at the base and then extending outwards along the veins of leaf edges. In severe conditions, branches start drying one after another in a sequence resulting in death of the whole tree (Khanzada et al., 2004). In Pakistan, infected trees die suddenly in their numbers and there is no end in sight (Al-Adawi et al., 2006). According to Masood et al. (2010), the infected leaves wither but usually remain attached to the dying tree. The mortality of the trees is usually by the blockage of xylem and phloem (vascular bundles) from proper flow of nutrients (Khuhro et al., 2005). The twigs and branches usually show internal discolouration. Brown streaks in vascular regions are visible upon splitting the twigs lengthwise (Narasimhudu and Reddy, 1992; Sharma, 1993; Ploetz et al., 1996; Khanzada et al., 2004). The conducting tissues are damaged, blocking the food supply from the leaves to roots and other parts, resulting in partial to full drying of the plant (Khuhro et al., 2005). Infected plants also show abundant gum secretion from uninjured branches, stem, and main trunk. Initially, the gum appears as a small droplet. However, as the disease progresses, it increases and covers most of the branch and trunk. Under severe conditions, the outer wood of a branch cracks and splits and exudes a yellow to brown, gum-like substance (Khanzada et al., 2004). On scrapping 14 University of Ghana http://ugspace.ug.edu.gh of severely infected trunk, it usually produces rotted cankers and in some cases oozing of bad smelled liquid (Masood et al., 2010). These symptoms described above may be found alone or in combination with two or more symptoms in different mango orchards on the same infected plant (Ploetz et al., 1996; Iqbal et al., 2007). Predisposing factors of mango trees to the mango tree decline disease Lasiodiplodia theobromae has been termed a weak pathogen, as its ability to infect host plants may be related to host predisposition resulting from environmental stress (Cilliers et al., 1993). Although fungi are the implicated inciters in many locations, abiotic stresses including host nutritional deficiencies are thought to play a role (Schaffer et al., 1988). Kazmi et al. (2005) showed that these abiotic factors include humidity, high temperature, sun scold, water stress and drought, unskilled cultural practices, less attention to infected plants by the farmers and growers also contribute to the development of quick decline disease. According to Khanzada et al. (2004), plants growing under water stress conditions in Pakistan show severe symptoms compared to frequently watered plants, indicating that water stress apparently predisposes the plants to disease and enhances the severity of this disease. Another factor identified is poor orchard management practices (Malik et al., 2005; Masood et al., 2011). Panhwar et al. (2007) observed that disease incidence increased in the stressed orchards due to deficiency of nutrients, shortage of water, irregularly monitored fields and having no preventive/cultural measures against the mango tree decline disease. During the field survey of this research work, it was noted that some farmers suspected that mechanical injuries by farm equipment and vehicles, and fire, predispose trees to the decline 15 University of Ghana http://ugspace.ug.edu.gh syndrome. This view point however, has not been confirmed by scientific evidence and therefore remains in the books as speculative. Incidence and severity of the mango tree decline disease The mango tree decline syndrome was recently recognized in virtually all mango-producing regions of the world (Ramos et al., 1991; Anwar et al., 2012). For instance, this decline syndrome is a recent severe threat to the Pakistan mango industry (Saeed et al., 2011). Incidence of this menace was found to be 20% and more than 60% in Punjab and Sindh Provinces of Pakistan respectively; and 60% in Al Batinah region of Oman (Al-Adawi et al., 2006; Saeed et al., 2006). This decline syndrome has also been reported in Brazil, Florida, USA and India (Verma and Singh, 1970; Ribeiro, 1980; Ploetz et al., 1996; Al-Adawi et al., 2006; Fateh et al., 2006; Saeed et al., 2008). Leghari (2005) isolated 12 species of fungi belonging to 10 different genera from infected mango trees showing sudden death syndrome symptoms, with 20.0-83.30% incidence and 62.5-85.0% severity of mango gummosis caused by Botryodiplodia theobromae and 56.7-73.3% incidence and 62.5-78.8% severity of mango decline caused by Fusarium solani. The decline syndrome has been conjectured to be present in Ghana, because of the similar symptoms found on diseased mango trees in the field. This necessitated the commencement of the present study, to confirm the identity and occurrence of the mango tree decline disease in Ghana. 16 University of Ghana http://ugspace.ug.edu.gh Economic importance of the mango tree decline disease Mango is known as the ‘king of all fruits’ (Sial, 2002; Litz, 1998). Mango is one of the most important fruit crops in the tropics, and approximately makes up 50% of tropical fruits produced in the world (Jedele et al., 2003). As aforementioned, it is grown in tropical regions of Pakistan, India, USA, Oman, China, Indonesia, Brazil, Mexico and Thailand, Nigeria, Kenya, Ghana, etc. The production, trade and consumption of mango fruits have increased significantly both domestically and internationally due to its attractive sensory properties and growing recognition of their nutritional and therapeutic properties (Bicas et al., 2011). Nonetheless, this fruit crop suffers from a number of diseases at various stages of its development. Among the various mango diseases is the mango tree decline syndrome which is of great economic importance. This disease causes death in plants at different stages of development, from seedlings to mature trees (Gallo et al., 2002). It poses both qualitative and quantitative losses of the crop. Infected trees may die within a very short time. In Pakistan, infected trees die suddenly in their numbers and there is no end in sight. According to a report prepared by the Ministry of Food, Livestock and Agriculture (Minfal) Pakistan, the disease is of great economic importance because matured mango plant dies within a short span of time. This disease was spreading in large areas in the two provinces first in the Punjab and recently in the Sindh (Anon., 2006). The same can be said of the incidence of the mango tree decline disease in Ghana having been reported in the Ashanti, Brong-Ahafo, Eastern, Greater-Accra, and Northern regions. However, the percentage incidence and mean severity are higher in the local mango variety farms than the exotic mango variety farms. 17 University of Ghana http://ugspace.ug.edu.gh Aetiology of mango tree decline disease Fungi as a causative pathogen Botryosphaeria ribis was the first pathogen to be reported as a primary cause of mango tree decline disease (Ramos et al., 1991). Many additional fungi have been associated with symptomatic tissues exhibiting bud necrosis, tip dieback, gummosis and vascular discolouration, including: Alternaria alternata, Cladosporium sp., Colletotrichum gloeosporioides, Dothiorella dominicana, Fusarium spp., Lasiodiplodia theobromae, Penicillium sp., Pestalotiopsis sp. and Phomopsis spp. (Ploetz et al., 1996). Jiskani (2002) reported that fungi such as Botryodiplodia theobromae, Alternaria, Acremonium, Scytalidium, Fusarium and Ceratocystis stained both the surface and the deeper wood of mango. Sharma (1993) established Botryodiplodia theobromae as a primary cause of dieback of mango. A year earlier, Narasimhudu and Reddy (1992) isolated Botryodiplodia theobromae from mango trees severely affected by gummosis and confirmed its pathogenicity. Recently, Ceratocystis fimbricata has been isolated from vascular bundles of declining trees and is considered to be one of the contributing factors of mango decline (Fateh et al., 2006). Jason et al. (2005) noted that mango tree decline is due to attack of mycoflora which infect vascular tissue of plant and ample anatomical modifications were expected and because of this fact, good yield is impeded. This pathogen also attacks ripe fruits in storage at the base of pedicle (stem end rot) and the circular brown area near the stem end further develops towards the lower portion of the fruit. Later, the entire fruit surface is covered with the dark brown to black area and complete fruit rotting occurs in 2-3 days. The disease may also start from injured portion on the fruit surface (Rawal and Ullasa, 1989; Johnson, 1998; Jiskani, 2002; Khuhro et al., 2005). 18 University of Ghana http://ugspace.ug.edu.gh Nematodes and insects as agents associated with the mango tree decline disease Nematodes including Criconemella sphaerocephala, Helicotylenchus dihystera, Hoplolaimus indicus, Hemicriconemoides mangiferae, Meloidogyne spp. and Rotylenchulus reniformis have been associated with the presence of mango decline syndrome (McSorley et al., 1980; Siddiqui, 2007). Two nematodes, Hemicriconemoides mangiferae and Xiphinema brevicolle have also been reported from declining mango trees in South Africa where these two nematodes caused decline of lychee, Litchi chinensis (Milne et al., 1971 and 1975). The most frequently found bark beetle on diseased tree was identified as Hypocryphalus mangiferae Stebbing (Coleoptera: Scolytidae). This insect preferred diseased or dried portions of wood and made tiny holes from which saw dust emitted, that was the characteristic damage pattern of this beetle. The beetle produces galleries in the cambium of affected trees, feeding primarily on fungi and also serving as a wounding agent which facilitates infection and transmission of the pathogens (Ploetz, 2003). Dieback and gummosis; typical symptoms of the mango tree decline disease Ahmed et al. (1995) reported that the onset of dieback becomes evident by discolouration and darkening of twigs from tip to downward due to Diplodia natalensis. Ploetz et al. (1997) observed the symptoms of decline, tip dieback and gummosis from mango nurseries artificially inoculated with Alternaria alternata, Glomerella cingulata, Dothiorella dominicana, Botryodiplodia theobromae and Phomopsis sp. Khanzada et al. (2004) and Saleem et al. (2006) showed that gum was the most common symptom and Lasiodiplodia theobromae was the most abundant isolated fungus, whereas, Fateh et al. (2006) found Ceratocystis fimbriata associated with gummosis. 19 University of Ghana http://ugspace.ug.edu.gh On the basis of the work done by the above researchers, it is conjectured that mango tree decline is a disease complex involving biotic and abiotic factors. Identification of Lasiodiplodia theobromae Lasiodiplodia theobromae is a fungal plant pathogen with a very wide host range including date palm (Phoenix dactylifera L.), Jatropha spp., Grapevine (Vitis vinifera L.), sappan wood (Caesalpinia sappan), Euphorbia ingens, and Theobromae cacao L. It is an Ascomycete in the family Botryosphaeriaceae, which causes rotting and dieback in most species it infects. It is a common post-harvest fungus disease of citrus known as stem-end rot. It is a cause of bot canker of grapevine (Úrbez-Torres et al., 2008) and has been implicated in the widespread mortality of baobab (Adansonia digitata) trees in Southern Africa. On rare occasions it has been found to cause fungal keratitis, lesions on nail and subcutaneous tissue of humans (Ellis, 2016). Though this fungus is commonly known as Botryodiplodia theobromae (Pat.), Sutton (1980) has adopted the name Lasiodiplodia theobromae Griff. and Maubl. as suggested by Zambettakis (1954). Lasiodiplodia theobromae has also been recognized by Hawksworth et al. (1995), and Botryodiplodia theobromae is now, therefore, considered to be a synonym of Lasiodiplodia theobromae. Other synonyms include Diplodia theobromae (Pat.) Nowell; Lasiodiplodia tubericola Ell. and Ev.; Diplodia tubericola (Ell. and Ev.) Taubenh; Botryodiplodia tubericola (Ell. and Ev.) Petrak. Teleomorph: Botryosphaeria rhodina (Berk. and Curt.); synonym: Physalospora rhodina Berk. and Curt. Khanzada et al., (2004) noted that on Potato Sucrose Agar (PSA), colonies are initially white, soon becoming black and fast-spreading with immersed and superficial, branched, septate mycelium. 20 University of Ghana http://ugspace.ug.edu.gh They also observed on the PSA shiny black pycnidia produced on the surface of the colony. Conidia were initially hyaline, unicellular, subovoid to ellipsoidal, with a granular content. At maturity, conidia became two-celled, cinnamon to dark brown, thick walled, ellipsoidal, often with longitudinal striations, 18-30 µm × 10-15 µm in size. They also observed that the morphological characters for pycnidia and conidia were similar to those given by Sutton (1980). On the other hand, 12 isolates of Lasiodiplodia theobromae were isolated from the infected fruits of different banana varieties collected from various localities of Tamil Nadu, India, by Sangeetha et al. (2010). These 12 isolates widely varied in their morphological and growth characters. The results showed that most of the L. theobromae isolates exhibited significant differences in morphology, colour and spore size. However, all 12 isolates invariably took 3–4 days to cover the 90 mm Petri plates, and they either produced grey or greyish white, or greyish black or black coloured colonies on PDA. However, after 2 weeks all the isolates turned black due to enormous sporulation. Conidia of all the isolates were ovoid to elongate. Ko et al. (2004) observed that the fungus on citrus produced greyish black colonies and black ostiolate pycnidia on V8 agar, and conidia were ovoid to elongated. Sangeetha et al. (2010) showed considerable variations in pycnidial production and in the formation of pycnidial stromata by these isolates. Some isolates produced matted hyphae forming stroma, which contained several pycnidia. Some were devoid of stroma and produced pinhead or mustard like pycnidia. In support of the observations, Woodward et al. (2005) observed L. theobromae infecting eggplant and stated that the fungus produced greyish colonies with aerial hyphae and black ostiolate pycnidia enmassed into stroma. Matured, two-celled spores were found only in 2 to 3 weeks old cultures. 21 University of Ghana http://ugspace.ug.edu.gh According to a morphological description by Ellis (2006), colonies of L. theobromae are greyish sepia to mouse grey to black, fluffy with abundant aerial mycelium; reverse fuscous to black. Pycnidia are simple or compound, often aggregated, stromatic, ostiolate, frequently setose, up to 5 mm wide. Conidiophores are hyaline, simple, sometimes septate, rarely branched cylindrical, arising from the inner layers of cells lining the pycnidial cavity. Conidiogenous cells are hyaline, simple, cylindrical to sub-obpyriform, holoblastic, annellidic. Conidia are initially unicellular, hyaline, granulose, sub-ovoid to ellipsoid-oblong, thick-walled, base truncate; mature conidia one-septate, cinnamon to fawn, often longitudinally striate, 20-30 x 10-15 µm. Classical microscopic features alone cannot be used to characterise the isolate since cultural morphology may vary widely on media and spore size vary significantly with environmental conditions. For instance, a study by Sangeetha et al. (2010) demonstrated the high degree of genetic variability within Lasiodiplodia theobromae isolates from banana. Presently, characterisation of isolates of plant pathogenic fungi based on DNA variability is considered useful. The case in point is the application of developed RAPD molecular markers in studies of genetic diversity which has been shown to be useful for several fungal pathogens (Williams et al. 1990; Maclean et al. 1993). By far, 20 species of Lasiodiplodia have been described and they are differentiated on the basis of conidial and paraphyses morphology. The more recently described species (described since 2004) have been separated not only on morphology, but also on the basis of ITS and EF-1α sequence data. Punithalingam (1976) included several of the species known at that time as synonyms of L. theobromae since he could not separate them on morphological characters. However, on account of 22 University of Ghana http://ugspace.ug.edu.gh its morphological variability and wide host range it seems likely that L. theobromae is a species complex. Recent studies based on sequence data have confirmed this and eight new species have been described since 2004 (Pavlic et al., 2004, 2008; Burgess et al., 2006; Damm et al., 2007; Alves et al., 2008). Species specific primers have been designed based primarily on the sequence dissimilarities of the ITS region of representative Lasiodiplodia species and have been used successfully and sequences have been used to characterize the pathogen from a wide range of crop plants. (Correl et al., 1993). Sequence analysis of the ITS region has also been used to accurately differentiate between and identify several species within the Lasiodiplodia genus. Species in the genus Lasiodiplodia have been distinguished based on their DNA phylogeny in association with conidial morphology and dimensions, and morphology and size of paraphyses. Burgess et al. (2006) used septation of pycnidial paraphyses to differentiate Lasiodiplodia species including L. crassispora, L. gonubiensis, L. rubropurpurea, L. theobromae and L. venezuelensis. However, this character needs to be interpreted carefully since paraphyses are aseptate when they are young but later they become septate. Damm et al. (2007) distinguished L. plurivora from L. crassispora and L. venezuelensis on the length and shape of the paraphyses. Burgess et al. (2006) used conidial dimensions to differentiate L. crassispora, L. rubropurpurea and L. venezuelensis from L. gonubiensis and L. theobromae. Furthermore, Alves et al. (2008) distinguished L. parva, and Pavlic et al. (2008) distinguished L. margaritacea from all other species on account of their small conidia. 23 University of Ghana http://ugspace.ug.edu.gh The use of traditional methods (morphology, colony colour, conidia size, pycnidial production, etc.) has not been satisfactory for differentiating between species, sub-species and between different isolates of pathogenic fungi. Thus molecular approaches have gained popularity (Sangeetha et al., 2010). It was therefore expedient to augment data obtained from the morphological studies on the Ghanaian Lasiodiplodia spp. with molecular data to complete the research findings. Control of the mango tree decline syndrome Many researchers have conducted various experiments on the diseases of mango plants and fruits and evaluated various ways by which the diseases can be controlled. It was suggested that integrated disease management practices must be applied by using different fungicides in combination with suitable insecticides; whereas different cultural practices may also help to control the diseases. On the other hand, it is also pertinent to mention that fungicides increase cost of crop production and their use may not be environmentally friendly and may pose health risks to farm workers. Fungicides may decrease the fruit quality because of their toxic substance residues which may remain in the fruits for a long time (Jiskani, 2002). The continual use of pesticides is frowned at by the environmentalists and consumers alike. A study by Arif et al. (2015) evaluated the effectiveness of plant activators used in conjunction with the fungicide thiophanate methyl in managing mango decline disease. The study was conducted in the Multan district using trees rated as 1-2 on a decline severity scale and displaying symptoms of gummosis, bark splitting, canker formation, and leaf drooping. Experimental treatments included 3 plant activators viz. Bion, Planofix, and Root king in conjunction with or without thiophanate methyl, delivered through a macro infusion system. After 3 months, thiophanate methyl, in combination with Bion, was found to be the most effective treatment with trees displaying no 24 University of Ghana http://ugspace.ug.edu.gh apparent disease symptoms. When thiophanate methyl was used alone, or in combination with Root king and Planofix, the symptoms of bark splitting and gummosis persisted. Dieback can also be managed by pruning of infected plant parts from 7-10 cm below the infection site and pasting the cut ends with clay mixed cow dung or Copper oxychloride or Bordeaux mixture. In cases of gummosis, diseased parts may be cleaned/removed and pasted either with Bordeaux or Copper oxychloride paste (Rawal and Ullasa, 1989; Johnson, 1998; Jiskani, 2002; Khuhro et al., 2005). In Ghana, many fungicides (such as Mancozeb, Funguran and Bendazim), both systemic and contact types, have been used for the management of field diseases of many economic crops. The nature of control measure practised in a particular mango farm depends on several factors with the major factor being the targeted market and the common practice being applied in a localized area. For instance, farms producing mainly for the local market do not usually carry out any control measure; many farms are unattended to and whatsoever fruits obtained during the season are sold on the local market. In contrast, farmers targeting the conventional fresh fruit market practice regular pruning with heavy fungicide applications (Honger et al., 2014). Again during the field visits, some farmers were found to be in the habit of spraying diseased seedlings and young plants showing vascular browning with fungicides every 2 weeks, until the symptoms ‘disappear’. This invariably increases the chemical load in soils, water bodies and the environment at large. Muhammed et al. (2005) reported that Carbendazin is effective in inhibiting the growth of mycelium of L. theobromae in vitro and field conditions suppressing gum exudation, dieback and wilting resulting in significant improvement in vegetative growth of plants. Sahi et al. (2012) showed that 25 University of Ghana http://ugspace.ug.edu.gh Mancozeb was slightly more effective in inhibiting mycelia growth of L. theobromae. Shelar et al. (1997) previously evaluated in vitro effectiveness of several fungicides namely Aureofungi, Carbendazin, Captan, Benomyl, Mancozeb, Copper oxychloride and thiophanate-methyl against L. theobromae using solid and liquid Richard’s media. They found that Benomyl (0.9%), Captan (0.2%), Carbendazin (0.1%), Mancozeb (0.25%) and thiophanate-methyl (0.19%) were highly effective in inhibiting L. theobromae in both solid and liquid media. Mahmood et al. (2002) on the other hand showed that foliar spray Topsin-M (Thiophanate-methyl) at 1 g/L reduced infestation of L. theobromae to 10% and two sprays of the same fungicide completely inhibited the fungus causing mango decline disease. Synthetic fungicides increase cost of crop production; their use may pose health risk to farm workers and consumers; pollute the environment; and decrease fruit quality when their toxic residues remain in fruits for a long time. These concerns have led to the re-examination and improvement of many old disease management practices used in controlling plant diseases (Agrios, 2005). Biological control using natural products presents to us, an alternative and a viable means of pests and diseases management. This method has been used for over six decades and has recorded many successes. Generally, plants contain many secondary metabolites with peculiar individual properties which differ from one plant species to another. Due to the variation in chemical constituents, plants differ in their antibacterial and/or antifungal activity. The yield of active compounds which may be obtained may differ, depending on the solvents and the methods of extraction. For instance, for over 30 years now, research in the Microbiology laboratory of the Department of Plant and Environmental Biology, University of Ghana, and elsewhere have shown that extracts from over 30 angiosperm 26 University of Ghana http://ugspace.ug.edu.gh plants can be used to control (or inhibit growth of) several pathogens such as Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Cladosporium cucumerinum, Colletotrichum eragrotidis, Fusarium verticilloides, Penicillium citrinum, Phytophthora palmivora and Sclerotium rolfsii (Osei, 1992; Odamtten and Okyere, 1994; Mensah, 2001; Hoffman et al., 2004; Frimpong, 2007; Wiafe-Kwagyan, 2007; Yasmin et al., 2008). Odebiyi (1986) indicated that aqueous extracts of Jatropha podagnea have antibacterial and antifungal activity against dermatophytic fungi such as Candida albicans, Epidermophyton floccosum, Microsporus canis, and Trichophyton rubrum. Saksena and Tripathi (1986) used plants such as Eucalyptus citricola, Chenopodium album, Cymbopogon flenosus and Thespisia populea to show their fungistatic effect against Mucor mucedo. Mensah (2001) observed that acetate and dichloromethane extracts of Azadirachta indica and Clausena anisata suppressed the vegetative growth and zoospore discharge of both Phytophthora megakarya and P. palmivora. Similarly, the petroleum ether seed extract of Griffornia simplicifolia significantly (p≤0.05) suppressed indirect germination of the sporangium of Phytophthora palmivora and P. megakarya at higher concentrations (Wiafe-Kwagyan, 2010; Wiafe-Kwagyan and Odamtten, 2013). Okigbo et al. (2009) demonstrated that water and ethanol extracts of Chromolaena odorata and Azadirachta indica leaves serve as a good natural plant fungicide (protectant) against yam tubers in storage against the yam rot spoilage fungi caused mostly by Fusarium oxysporum, Aspergillus niger and Lasiodiplodia theobromae. However, Azadirachta indica was found to be more potent. A recent review by Uyi et al. (2014) indicated the ethno-pharmacological, fungicidal, nematicidal importance of Chromoleana odorata and its use as a fallow species and as a soil fertility improvement plant in 27 University of Ghana http://ugspace.ug.edu.gh the slash and burn rotation system of agriculture. This has contributed to its continued use and spread in Nigeria. Odamtten (1992) demonstrated in his studies that C. odorata aqueous leaf extract can be used to control Fusarium verticilliodes (F. moniliforme) infecting some economic crops. Leaves and seeds of Carica papaya L. are known to contain proteolytic enzymes (papain, chymopapain), alkaloids (carpain, carpasemine), sulfurous compounds (benzyl isothiocyanate), flavonoids, triterpenes, organic acids and oils (Osuna-Torres et al., 2005). Extracts from different papaya tissues have been shown to be bioactive. Aqueous extracts of the leaves and seeds are known to have antifungal activity against Colletotrichum gloeosporioides (Bautista-Baños et al., 2002). Recent studies by El-Zaher (2014) showed that aqueous extract of C. papaya seed has inhibitory activity against Aspergillus flavus with inhibition zones ranging between 11-16 mm. Recent research in the Laboratory of the Microbiology Section of the Department of Plant and Environmental Biology has shown that biotoxins from the aqueous extract of Plectranthus coleoides depressed vegetative growth of Lasiodiplodia theobromae at 1:1 v/v dilution by nearly 50% and prevented sporulation and pycnidia formation (Agyemang-Boateng, 2016). The environmental detrimental consequences of continual use of pesticides in our ecosystem informed the search for alternative bio-pesticides in plants for the control of plant pathogenic fungi. In this thesis, this quest is reflected in the in vitro studies of the effect the aqueous extract of three plants, namely, Chromolaena odorata (siawa weed), Azadirachta indica (neem) and Carica papaya (pawpaw) on the vegetative growth and sporulation of L. theobromae causing the mango tree decline disease in Ghana with the view to building up information of a biocontrol using biotoxins from natural sources. The bioactive compounds coming from natural sources are presumed to be 28 University of Ghana http://ugspace.ug.edu.gh biodegradable in control contrast with chemical from fungicides which are not environmentally friendly. 29 University of Ghana http://ugspace.ug.edu.gh CHAPTER 3 MATERIALS AND GENERAL METHODS Field survey of exotic and local mango variety farms Study area The field survey was carried out in randomly selected farms within four agro-ecological zones encompassing five administrative regions of Ghana, namely the Ashanti, Brong-Ahafo, Eastern, Greater-Accra and Northern Regions. At least, two districts were visited and surveyed in each of these five administrative regions. The four agro-ecological zones were the coastal savanna, guinea savanna, semi-deciduous and transition zones. Field visits A total of 36 farms, comprising 18 each of exotic and local mango variety farms, were visited and studied to ascertain the nature of the mango decline disease in the field. Interviews sessions were held with the farmers and questionnaires administered in search of information. Finally, both symptomatic (diseased) and healthy plant parts were sampled and sent to the Microbiology laboratory of the Department of Plant and Environmental Biology (formerly Department of Botany), for further studies. The questionnaires administered to the farmers are in Appendix 1. Assessment of the mango tree decline disease and its symptoms In the months of June, July and August, 2015, field surveys were carried out to 36 farms across Ghana, to ascertain the symptoms and the extent of the mango decline syndrome in the country. 30 University of Ghana http://ugspace.ug.edu.gh Farms in four of the five agro-ecological zones were visited namely the coastal savanna, guinea savanna, semi-deciduous and transition agro-ecological zones. The survey was to assess the nature of the disease symptoms; distribution of the disease in the country; and the appearance or non-appearance of decline disease on the visited mango farms. Also during the period of visits to the farms, leaves, twigs, fruits, branches and bark of the mango trees were inspected for the presence of the disease symptoms. The possible difference and similarity of the disease symptoms on both exotic and local crop varieties were examined. When required, farmers were interviewed and the reasons for the expression of symptoms were deduced. Branches, stems and twigs were sliced, opened or scraped in the field to aid the detection of disease symptoms. Tree barks were also removed for the assessment of symptoms. Interaction with farmers During the field survey, farmers, farm managers and farmers’ groups were interviewed on the incidence, severity and symptoms of the mango decline disease on their farms. Questionnaires were also administered to farmers and specific answers on their knowledge on the prevalence of the disease were obtained. The responses from all the visited farmers were recorded for analysis. Field survey for the disease incidence and severity in Ghana In the months of June, July and August, 2015, field surveys were carried out on 36 farms to ascertain the incidence and severity of mango decline disease in Ghana. The exotic mango variety farms (size > 1 acre), as well as cluster local variety mango trees were selected randomly. Disease incidence and severity were evaluated with the aid of a modified model proposed by Cardoso et al. (2004) for evaluating disease incidence and severity of cashew gummosis caused by L. theobromae. 31 University of Ghana http://ugspace.ug.edu.gh Incidence and severity were assessed at the same time using the modified method of Cardoso et al. (2004). On each farm, 10 trees of the Keitt or Kent variety (the only varieties most common to all farmers) were randomly sampled. Similarly, 10 local variety mango trees in cluster were sampled at random. Incidence (I) was based on the presence of typical symptoms of the disease. Disease incidence (I) was calculated by the equation: I = ∑ (x/N) Where x = number of diseased plants N= total number of plants evaluated. Severity (S) was estimated by the equation: S = ∑(xini)/n Where xi= disease grade (modified) as per Table 1 ni= number of diseased plants on the ith grade of the disease scale n = total number of diseased plants evaluated. 32 University of Ghana http://ugspace.ug.edu.gh Table 1. Disease severity rating scale used for the assessment of disease severity in different mango farms in Ghana Rating Meaning 0 No symptoms 1 Little wilting of upper tips 2 Wilting and small exudates of gum, yellowing of leaves, little browning of vascular tissues 3 Drying of branches, heavy gum exudation from branches, large scale browning of vascular tissues 4 Splitting of the bark, gum exudation from branches as well as from main trunk, drying of more than half of the tree 5 Death of plant Cardoso et al., 2004 (modified) 33 University of Ghana http://ugspace.ug.edu.gh Incidence and severity of mango tree decline disease in Ghana in 2015 Incidence and severity of mango tree decline disease in five administrative regions of Ghana in 2015 The survey was carried out in five out of the ten administrative regions of Ghana comprising Ashanti, Brong-Ahafo, Eastern, Greater-Accra, and Northern Regions. The results obtained from the survey on each farm in the same administrative region was pooled for analysis. Incidence and severity of mango tree decline disease in four agro-ecological zones of Ghana in 2015 The results obtained from each surveyed farm on the disease incidence and severity in the same agro-ecological zone was composited for further analysis. The four agro-ecological zones visited were the coastal savanna, guinea savanna, semi-deciduous and transition zones. Materials Materials used in preparing culture media Plant materials namely local mango variety leaves, bark and fruits; exotic (Kent) mango variety leaves, bark and fruits; Pseudotsuga pine needles; Egyptian date palm (Phoenix sp.) seeds; and soil, were collected during the months of June to December, 2015. The mango plant parts and fruits were collected from farms in the country and composited. The pine needles and soil were collected from the University of Ghana campus. The Egyptian date palm fruits were imported from Egypt, and the seeds subsequently removed. The identities of these plants were confirmed at the University of Ghana Herbarium, Department of Plant and Environmental Biology (formerly Department of Botany). 34 University of Ghana http://ugspace.ug.edu.gh Influence of biotoxins in three plants on in vitro vegetative growth of L. theobromae Materials used in preparing the aqueous extracts of plant materials used in in vitro studies on the control of the pathogen Plant materials namely Chromolaena odorata (locally called Acheampong weed) leaves, Azadirachta indica (neem) leaves and Carica papaya (pawpaw) seeds were used in the study. These were collected during the months of August to October, 2015. They were obtained from the University of Ghana campus and its surrounding suburbs; their identities were confirmed at the University of Ghana Herbarium, Department of Plant and Environmental Biology, University of Ghana, Legon. Preparation of extracts Preparation of aqueous extracts of plant materials Leaves and bark of local and exotic mango varieties, Egyptian date palm (Phoenix sp.) seeds and Psuedotsuga spp. pine needles were fragmented into pieces and air dried for 7 days. An amount of 1 kg each of the dried plant materials was weighed and pulverised into powder using a commercial milling machine (Fritsch Company, Germany). Exactly 110 g of the powdered samples was placed in 1 L Erlenmeyer (conical) flask and was done in triplicate; 800 ml each of distilled water was poured into the three conical flasks. Aqueous extract was prepared for each plant material by the hot percolation method using electric hot plate at a temperature of 60 ºC for a period of 2 hr. The supernatant was then filtered using filtration apparatus. The extracts were finally reduced to dryness at 60 ºC using a rotor evaporator (Büchi, Switzerland). The remaining traces of solvent in the extracts were removed by keeping the extracts in an oven and then in desiccators. 35 University of Ghana http://ugspace.ug.edu.gh Preparation of ethyl acetate extracts of plant materials A similar procedure as described in Section 3.4.1 was used except that ethyl acetate was used as the extraction solvent. Exactly 110 g of the powdered plant materials was placed in 1 L conical flask; 800 ml of ethyl acetate solvent was poured into the conical flasks containing the plant material. This was done in triplicate. Extraction was carried out for each plant material using the hot percolation method with a water bath as a source of heat at a temperature of 60ºC for a period of 2 hr. The supernatant was then filtered using filtration apparatus. The extracts were finally reduced to dryness at 60ºC using Rotor evaporator (Büchi, Switzerland). The remaining traces of solvents in the extract were removed by keeping the extracts in an oven and then desiccators. Preparation of petroleum ether extracts of plant materials The same process was repeated as in the case of the ethyl acetate above (Section 3.4.2), except that petroleum ether was used as the extraction solvent. Preparation of soil extracts Exactly 20 g of the soil was suspended in 200 ml distilled water, and the mixture thoroughly mixed by stirring and swirling. Following a modified protocol described by Subba Rao (1977), an amount of 17.75 g of the filtrate was measured to obtain a soil extract to be used subsequently for media preparation. Preparation of mango fruit juice extracts Exactly 200 g of the pulp of ripe mango fruits was weighed. The pulp was placed in a sterile white muslin cloth and squeezed gently to obtain the fruit juice. The required volume of the extracted mango juice was measured for media preparation. 36 University of Ghana http://ugspace.ug.edu.gh Preparation of aqueous extracts of plant materials for in vitro control cultures Chromolaena odorata (Acheampong) leaves, Azadirachta indica (neem) leaves and Carica papaya (pawpaw) seeds were fragmented into pieces and air dried for 7 days. An amount of 1 kg each of the dried plant materials was weighed and pulverised into powder using a commercial milling machine (Fritsch Company, Germany). Exactly 200 g of each of the powdered samples was weighed and transferred into 1 L conical flask; 1 L each of distilled water was poured into the three conical flasks, and allowed to stand for 12 hr. The supernatant was then strained through a clean white muslin cloth. Media composition and preparation Commercially formulated synthetic media Synthetic media used were Potato Dextrose Agar (PDA), Czapek-Dox Agar (CDA), dichloran rose- bengal chloramphenicol agar (DRBC), oxytetracycline glucose yeast extract agar (OGYE), water agar (WA), Potato Dextrose Broth (PDB), Czapek-Dox Broth (CDB), and oxytetracycline glucose yeast extract broth (OGYE-B). PDA, CDA, DRBC, OGYE and WA were used for the preparation of solid media at the rates of 39 g/L, 50 g/L, 31.5 g/L, 37 g/L, and 20 g/L respectively. The liquid media (broth) were prepared with PDB, CDB and OGYE in distilled water at the rates of 24 g/L, 35 g/L, 16.5 g/L, and 22 g/L respectively. The required amounts of the already formulated synthetic media were weighed and suspended in 1 L distilled water. Mixture was heated to boiling to dissolve the medium completely, and sterilized by autoclaving at 121 ºC and 1.1 kg/cm3 pressure for 15 minutes. 37 University of Ghana http://ugspace.ug.edu.gh Natural media prepared with extracts of plant materials The aqueous, ethyl acetate and petroleum ether extracts of the plant materials aforementioned (Section 3.4) were used in growth media preparation. The plant materials were local mango variety leaves and bark; exotic (Kent) mango leaves and bark; pine needles; and Egyptian date palm seeds. An amount of 20 g glucose was added to each of the powdered extracts in 1 L distilled water to obtain the growth media. 15 g of agar was added to obtain solid media for plate cultures. For liquid media, no agar was added. Media was sterilized by autoclaving at 121 ºC and 1.1 kg/cm3 pressure for 15 minutes. Natural media prepared with soil extract An exact amount of 17.7 g of the soil extract, 20 g glucose, 0.5 g Dipotassium phosphate and 15 g agar in 1 L distilled water, was formulated, heated to boiling and autoclaved at 121ºC and 1.1 kg/cm3 pressure for 15 minutes. Same protocol was followed to prepare soil extract broth but no agar was added. Natural media prepared with mango fruit juice In preparing the fruit juice extracted solid media, 15 g agar was suspended in 1 L extracted fruit juice, heated to dissolve the agar completely and autoclaved. No glucose was added. In preparing the broth, 0.5 L fruit juice was suspended in 0.5 L distilled water and autoclaved. Neither agar nor glucose was added to the broth. Double strength Potato Dextrose Broth (DPDB) Potato tubers were washed, peeled and cut into smaller pieces. Exactly 400 g of peeled potatoes were boiled in 500 ml of distilled water for 10-15 minutes, until it softened. The water was strained using 38 University of Ghana http://ugspace.ug.edu.gh clean white muslin cloth, the filtrate made up to 1 L, with distilled water, then 40 g glucose was added. The prepared medium was autoclaved at 121 ºC and 1.1 kg/cm pressure for 15 minutes. When needed, 30 g of agar was added to obtain double strength Potato Dextrose Agar. Potato Dextrose Agar (PDA) Potato tubers were washed, peeled and cut into smaller pieces. 200 g of sliced potatoes were boiled in 500 ml of distilled water for 10-15 minutes until it softened. The water was strained using clean white muslin cloth into 1000 ml beaker. Exactly 20 g glucose and 15 g agar were added to the supernatant in the beaker, then made up to 1000 ml with distilled water. The prepared medium was autoclaved at 121 ºC and 1.1 kg/cm3 pressure for 15 minutes. General methods Maintenance of stock cultures Stock cultures of the isolated fungus, Lasiodiplodia theobromae, were grown on 90 mm Petri dishes. The cultures were stored in incubators at 28±1 ⁰C and were sub-cultured every 3 weeks. Method of inoculation For both solid and liquid cultures, an inoculum of 4 mm agar discs obtained from the growing edge of the culture on PDA was used throughout. The number of replicates in each preparation was three. Measurement of pH The pH readings of culture media and filtrates were obtained using TOA pH HM-60 (Ogawa Seiki Co. Ltd. Japan). Readings of pH were made of autoclaved medium just before inoculation and at the end of the experiment at room temperature. 39 University of Ghana http://ugspace.ug.edu.gh Assessment of growth Fungal cultures Growth of cultures of the isolated fungus, Lasiodiplodia theobromae, on solid media in Petri dishes was assessed by measuring the diameter of cultures along two diameters drawn at the bottom of the Petri dishes, through the centre of the inoculated disc. Mean of the two measured diameters was calculated and used to determine radial growth rate. Growth in liquid cultures was assessed by estimating the dry weight of the harvested mycelia mat at the end of the incubation period. Mycelia collected on a previously weighed and dried Whatman No. 1 filter paper was dried at 75 ⁰C for 24 hr. The filter paper carrying the dried mycelia was then weighed, after it has been allowed to cool in a desiccator to obtain a constant dry weight. The dried weight of the mycelia was obtained by the difference in weight between the filter paper alone and the filter paper carrying the dried mycelia. Isolation of the causal agent Leaves, twigs, mango fruits and bark showing mango decline symptoms were sampled and collected from randomly selected farms and sent to the Microbiology laboratory of Department of Plant and Environmental Biology, for isolation of the causal agents. The isolation of causal agent was first done on water agar (WA) and then on Potato Dextrose Agar (PDA). WA (15 g/L) and PDA (39 g/L) were prepared and each mixture was autoclaved, allowed to cool and poured into clean sterilized plates and allowed to set (solidify). Pieces (1-2 cm) of the sampled plant parts (leaves, twigs, mango fruits, bark, vascular tissues) were taken from the advancing edge of infection with a sterile scalpel. These excised tissues were then surface sterilized 40 University of Ghana http://ugspace.ug.edu.gh in a 1% Sodium hypochlorite for 5 minutes, rinsed twice in sterile distilled water and blotted dry using a sterile tissue paper. These were plated singly on water agar plates and incubated till enough growth of the pathogen was observed. The growth were then sub-cultured on PDA and incubated till sufficient growth were observed. Pathogenicity test The pathogenicity test was carried out on ~10 months old, 9 potted exotic (Kent) mango variety and 9 local mango variety seedlings, obtained from Kpong (certified seedlings grower). All the exotic mango seedlings were grafted on local mango variety stock. This experiment was set up in Completely Randomized block Design (CRBD) in a screened house at the Department of Plant and Environmental Biology. Test plants were inoculated artificially by cutting a flap on the basal portion of the stem using a sterilized knife and inserting a 3 mm agar disc obtained from the advancing zone of the test fungus in Petri plates. With the grafted exotic mango seedlings, flaps were cut above the root stock. In the control trials, plants were inoculated with plain sterile agar disc without the test fungus. The inoculated portions were wrapped with Parafilm (a plastic paraffin film). Plants were irrigated after inoculation and the wrapping material was removed from the stems after 2 weeks of inoculation. Plants were then monitored for possible developments of the decline syndrome symptoms for 2 months. The pathogen was re-isolated from the test plants to confirm the pathogenicity. Disease incidence and severity in symptomatic plants were determined with aid of the modified disease hedonic score scale (Cardoso et al., 2004). 41 University of Ghana http://ugspace.ug.edu.gh Characterisation of the isolated causal agent Cultural, morphological and physiological characterisation Radial growth rate of isolated pathogen on solid media Synthetic media (CDA, DRBC, OGYE and PDA); plant extracts media (mango leaves and bark; Psuedotsuga pine needles; Phoenix Egyptian date palm seeds); and mango fruit juice media, in Petri plates, were inoculated with 4mm mycelia discs taken from advancing zones of the fungal culture. The discs were placed in the centre of the Petri plates. Using a ruler, the diameter of the advancing colony was measured after every 6 h on the reverse side of the plate along lines ruled across the diameter of the Petri plates. The inoculated plates were incubated at 28±1⁰Cunder three different light regimes: under continuous light; under continuous darkness; and under 12 h alternating light and darkness. The compacted fluorescent light was of 75 lux intensity. This was done in triplicate for each treatment. For each plate, the colony diameter was measured at 6 h intervals up to the 72nd h (3rd day). Mean growth rates were subsequently calculated. Vegetative growth and sporulation of the fungus in liquid media Exactly 30 ml each of the prepared liquid media was measured and transferred into 250 ml conical flasks. The flasks containing the media were inoculated with 4mm mycelia discs taken from advancing zones of culture PDA plates. The flasks were incubated at 28±1⁰C under three different light conditions (namely, under continuous light; under continuous darkness; and under 12 h alternating light and darkness) for 4 days. There were 3 replicates for each medium. Vegetative growth of the fungus was assessed by the conventional mycelial dry weight method. Mycelia mat was harvested after the required incubation period with pre-weighed dried Whatman 42 University of Ghana http://ugspace.ug.edu.gh No. 1 filter papers mounted in funnels. The filter papers with the harvested mat were dried at 75⁰C for 24 h and cooled in a desiccator to obtain a constant dry weight. The dry weight of the mycelia was obtained by difference in weight between the filter paper alone and the filter paper carrying the dried mycelia mat. Production of spores (sporulation) by the fungus after the incubation period was assessed by taking a piece of harvested mycelia mat. It was immediately mounted in lactophenol and observed under high power light microscope. The number of spores counted per 20-25 microscope field views was recorded. The initial pH of the media before inoculation was recorded and pH of culture filtrate after harvesting the mat was also recorded. Colour change of colony of the isolated pathogen during growth Colony colour change of the pathogen on solid media The synthetic and natural media with agar were poured into sterile Petri plates and allowed to set. Plates were inoculated with 4mm mycelia discs of the pathogen and incubated at 28±1⁰C under the three different light conditions, namely under continuous light; under continuous darkness; and under alternating 12 h light and 12 h darkness. Changes in the colour of the colony were monitored and observations were noted to aid in the identification of the pathogen. The colour change was monitored up to the 25th day of growth. Morphology of mycelia colony of the pathogen on solid media Petri plates with solid media were inoculated with 4 mm mycelia discs of the test fungus, and incubated at 28±1⁰C under different light conditions, namely under continuous light; under continuous darkness; and under alternating 12 h light and 12 h darkness. Changes in colony 43 University of Ghana http://ugspace.ug.edu.gh morphological characteristics namely, the form, elevation and margin of the colony were monitored for 15 days and observations noted. Plates were turned on their sides and upside to facilitate the assessment. 44 University of Ghana http://ugspace.ug.edu.gh Fig. 1. Key: Description of colony morphology (Leung and Liu, 2016). 45 University of Ghana http://ugspace.ug.edu.gh Morphology of the hyphae of the isolated pathogen Morphology of hyphae on solid media Plates with solid media were inoculated with the isolated fungus, incubated at 28±1⁰C and under the three different light regimes. To assess the morphology of the hyphae, a small portion of mycelia was mounted in lactophenol and observed under high power light microscope. This monitoring was done daily for 21 days. The colour, shape and size of hyphae; onset of septation; average number of septa per hyphal length; formation of chlamydospores and chlamydospores-like structures, and their sizes, were observed and recorded to help in the identification and characterization of the fungus. All micrographs were viewed under x400 high power magnification, otherwise, it is specified in the appropriate part of the thesis. Morphology of hyphae in liquid media The fungus was incubated in liquid media in 250 ml Erlenmeyer flasks to assess the morphology of the hyphae in liquid growth media vs. on solid media. A small portion of harvested mycelia mat was mounted in lactophenol and observed under high power light microscope immediately after harvesting the mycelia. The fungus was incubated for 4 days. Sporulation of the isolated pathogen on solid media The fungus was incubated at 28±1⁰C on all the selected solid media for 25 days under the three different light regimes (namely, under continuous light; under continuous darkness; and under 12 h alternating light and darkness). Sporulation (formation of spore/conidia) was checked daily after the 5th day of growth. For the assessment and estimation of sporulation, a small portion of mycelia was 46 University of Ghana http://ugspace.ug.edu.gh mounted in lactophenol and observed under high power light microscope. The number of spores per 20-25 microscope field views was counted and recorded. Morphological characteristics of conidia (spores) Shape, septation and striations on detected spores under high power light microscope were noted. The length and breadth (dimension) of 15-20 randomly selected conidia were measured with the aid of an eye piece graticule and a stage micrometer and then recorded to aid in the identification and characterization of the organism. The colour of the conidia was also noted. Molecular characterisation of the isolated causative pathogen The identification and characterisation of the isolated fungus was done using conventional methods namely cultural and morphological methods. The obtained results were further confirmed using polymerase chain reaction (PCR) with species specific primers and universal primers. Deoxyribonucleic acid (DNA) extraction The extraction of DNA from the isolated pathogen was carried out in the Biotechnology Laboratory of the College of Agriculture and Consumer Sciences of the University of Ghana. A plug of the isolated pathogen was plated on PDA and allowed to grow for 5 days. Using a sterile loop, the mycelia of the pathogen was scrapped from the surface of PDA and dried overnight in the laminar flow hood. The dried mycelia were mashed using mortar and pestle under liquid nitrogen after which the powdered mycelia were poured into micro-centrifuge tube. The DNA extraction was performed using the Sigma’s GenFlute Plant Genomic DNA Miniprep Kit following the manufacturer’s instructions. Exactly 350 µl of Lysis solution part A and 50 µl of Lysis solution B were added to the mycelia in the micro-centrifuge tube after which a pestle was used to 47 University of Ghana http://ugspace.ug.edu.gh gently mash the mycelia in the solution. The mixture was vortexed and incubated at 65°C for 10 minutes. Afterwards, 130 µl of precipitation solution was added to the mixture and mixed thoroughly and then placed on ice for 5 minutes after which the sample was centrifuged at 12000 x g for 5 minutes. The supernatant was pipetted onto a Genflute filtration column and centrifuged at 12000 x g for 1 minute after which the filtration column was discarded and 70 µl of binding solution was added to the flow through liquid in the collection tube and the mixture mixed thoroughly by inversion. A binding column was then prepared by pipetting 500 µl of the column preparation solution to a miniprep column and centrifuged at 1200 x g for 30 seconds, after which the flow through liquid was discarded. After that, 700 µl of the flow through liquid in the collection tube obtained prior to the preparation of the binding column was pipetted onto the prepared column and was centrifuged at 12000 x g for 1 minute after which the flow through liquid was discarded and the collection tube retained. The column was returned to the collection tube and the rest of the lysate prepared prior to column preparation, was added and the solution was centrifuged at 1200 x g for 1 minute after which the flow through liquid and collection tube were discarded while the column was retained. The binding column was placed in a clean 2 mL collection tube after which 500 µl of alcohol-diluted Wash Solution was added to the column and the solution centrifuged at 1200 x g for 1 minute after which the flow through liquid was discarded and the collection tube retained. Another 500 µl of the diluted Wash Solution was added to the column and centrifuged at 1200 x g for 3 minutes after which the binding column was transferred into a fresh 2 ml collection tube. 100 µl of pre-warmed (65°C) Elution Solution was added to the column and centrifuged at 1200 x g for 1 minute. The elution was repeated and the eluate containing the pure genomic DNA was collected and stored at -20°C till needed for the next part of the trial. 48 University of Ghana http://ugspace.ug.edu.gh Agarose gel electrophoresis, loading dye preparation and DNA ladder and primer reconstitution Agarose gel (0.8% w/v) was prepared by weighing 0.912 g of hydrated Molecular Biology agarose (BIORON, Germany) into a 114 mL 1 x TAE buffer. The mixture was weighed and heated in a microwave oven and allowed to cool to about 50°C after which 2.5 µL of Ethidium bromide (10 µg) was added and the flask swirled to ensure that the Ethidium bromide mix thoroughly with the agarose solution. The resultant solution was poured into a horizontal electrophoresis tray that has been mounted in a gel casting tray fitted with 10 teeth comb. The preparation was allowed to stand to enable the solution to polymerise after which the tray was removed from the gel caster and placed in an electrophoresis tank containing 1 X TAE buffer. Fresh loading dye was prepared by mixing 0.5 g of 0.25% bromophenol blue, 0.5 g of 0.25% xylene FF and 6 mL of 30% glycerol to 20 mL of ultra-pure water. To aid dissolution of the reagents in the water, few drops of 0.5 M EDTA were added and the mixture agitated carefully after which the preparation was stored till needed. A 1.0 kb DNA ladder was constituted by mixing 10 µL of stock solution, 20 µL of loading dye and 170 µL of sterile distilled water and the preparation stored until needed to be used. Primers were reconstituted into 100 picomoles by adding sterile distilled water and re-suspending overnight. This was also stored till needed to be used. Quality of extracted DNA was assessed by loading 5 µL of DNA into each well and electrophoresis carried out at 45 volts for 30 minutes. 49 University of Ghana http://ugspace.ug.edu.gh Polymerase chain reactions (PCR) The extracted DNA from the isolates of the pathogen was used as templates in polymerase chain reaction with the species specific primer Lt347-F (AACGTACCTCTGTTGCTTTGGC) and Lt347- R (TACTACGCTTGAGGGCTGAACA), specific to Lasiodiplodia theobromae. A second set of PCR was performed using the universal primer pair ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) to amplify the internal transcribed spacer (ITS) region of the isolates. PCR was carried out in a total reaction volume of 50 uL. The reaction mixture was made up of 34.25 uL of double distilled water, 5 uL of 10X PCR buffer (Invitrogen, Carlsbad, CA), 2.5 uL of deoxynucleoside-triphosphate mix (2.5 mM each), 0.25 uL bovine serum albumin (20 mg/ml), 2 uL each of the forward and reverse primer, and 0.2 uL of taq polymerase, 1.8 uL of magnesium chloride (50 mM) and 2 uL target DNA. The reaction was carried out in a Thermo Hybaid PXE Thermal Cycler. The reaction cycles were denaturing for 2 minutes at 94°C followed by 35 cycles of 1 minute at 94°C, 1 minute at 55°C, 2 minutes at 72°C and a final of 10 minutes at 72°C. Amplification products were separated by 1.5% w/v agarose gel stained with Ethidium bromide or gel red alongside 1.0 kb marker at 80 V for about 1 hour. Bands were observed under UV light and Polaroid photographs were taken using the Gene Flash Documentation System (Snygene Bio Imaging). Purification and sequencing of amplified product of the ITS region The PCR amplified product of the ITS region were sent to ETON Bioscience Laboratory at Raleigh in North Carolina for purification and sequencing. 10 picomole of each primer was used to sequence the product directly from both directions. 50 University of Ghana http://ugspace.ug.edu.gh Phylogenetic analysis The sequences of the internal transcribed spacer (ITS) region of a total of 23 isolates were used in the phylogenetic studies. These were made up of 8 isolates from the symptomatic mango tree bark and 15 sequences of ex-types and isolates of confirmed identities (downloaded from EMBL database) (Appendix 19). Included in the downloaded sequences was that of Botryodiplodia obtusa, which was used as the out-group. The sequences of the different gene regions of the isolates were aligned using Clustal W and the multiple sequence alignment obtained was used in a phylogenetic analysis using MEGA 5 software (Tamura et al., 2011). The Neighbour Joining (NJ) analysis was performed to infer the evolutionary history. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the data set (complete deletion option). Clade stability of the tree resulting from Neighbour Joining analysis was assessed by bootstrap analysis with 1000 replicates (Felsenstein, 1985) (Fig. 11). In vitro control of L. theobromae with aqueous extracts of selected plant materials The potency of aqueous extracts of selected plants (namely, Chromolaena odorata (Acheampong) leaves, Azadirachta indica (neem) leaves and Carica papaya (pawpaw) seeds ) in inhibiting the growth of the pathogen was tested. Growth of the fungus was assessed on solid and in liquid Potato Agar and Broth amended with varying concentrations (1:1, 1:2, 1:5 and 1:10 v/v dilutions) of the extracts. A 4 mm mycelia disc taken from advancing zone of L. theobromae culture was placed in the centre of the Petri plates, and in the flask containing the amended media of the appropriate 51 University of Ghana http://ugspace.ug.edu.gh dilutions in triplicates. Control plates and flasks containing extract-free media were also inoculated with the fungus. Radial growth on amended solid media with the extracts was assessed by measuring the diameter of cultures along two diameters drawn at the bottom of the Petri dishes, through the centre of the inoculated disc. Similarly, growth of the fungus in amended liquid cultures was assessed by estimating the dry weight of the harvested mycelia by the oven dry weight method. A plant extract was considered effective when it was able to suppress the growth of the pathogen on Petri plates and in flasks. Statistical analysis Subject matters considered in the survey questionnaires were collated and the data were subjected to Descriptive Statistics of Analysis Using statistical Package for Social Sciences (SPSS version 20). Experimental precautions 1. Sampled plant materials and glassware were carefully labeled to avoid misidentification. 2. All working areas were wiped with 70% alcohol before use. 3. The laminar flow cabinet was switched on for about 15 minutes before use. 4. Dried filter papers were kept in desiccators immediately they were removed from the oven to prevent re-absorption of moisture from the environment. 5. The filter papers were kept in an oven at a temperature of 60ºC in order to avoid burning of the filter papers and the mycelia. 52 University of Ghana http://ugspace.ug.edu.gh 6. Erlenmeyer (conical) flasks used in the experiment were covered with non-absorbent cotton wool plug. 7. The opening edge of medicinal bottles and Erlenmeyer flasks containing the media were flamed sterilized before each inoculation was done. 8. Inoculations needles (pins), cork borer and forceps were kept in 100% absolute ethanol and after that, were sterilized by flaming them over ethanol glass burners till they appeared red hot. 53 University of Ghana http://ugspace.ug.edu.gh CHAPTER 4 EXPERIMENTAL PROCEDURE EXPERIMENT 1 Field survey to assess the mango tree decline disease in the Administrative Regions of Ghana in 2015 With the reports of the mango tree decline syndrome in Ghana, it was necessary to evaluate the extent of the disease nationwide, particularly in the mango producing districts in the Administrative regions of Ghana. Selected farms within four agro-ecological zones of Ghana, namely, Coastal savanna, Semi- deciduous forest, Transitional, and Guinea savanna in the Administrative regions of Ashanti, Brong- Ahafo, Eastern, Greater-Accra, and Northern, were surveyed. Field visits were made to 36 farms comprising each of exotic and local mango variety farms. The method of administering the structured questionnaires is spelled out in the Materials and General Methods section. The following data were collected for exotic and local varieties of mango plants: typical symptoms of the disease, possible factors promoting symptoms expression in the field, and knowledge of the farmers on the decline syndrome. Results are presented in Plates 2-4; Figs. 2 & 3. 54 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 2 Disease incidence and severity of the mango tree decline disease in the Administrative regions of Ghana in 2015 In June, July and August of 2015, follow up survey were carried out on 36 farms in the 5 selected Administrative regions to quantify the incidence and severity of the mango tree decline syndrome in Ghana. This was necessary to ascertain the economic impact of the disease which is taking a heavy toll of the crop in the field. Disease incidence and severity were evaluated quantitatively for both local and exotic varieties of mango, using a modified method of Cardoso et al. (1998; 2004) (see Materials and General Methods section). Results obtained are summarized in Map 1, Table 2 and Appendices 2a & b. EXPERIMENT 3 Disease incidence and severity of the mango tree decline disease in the different agro-ecological zones of Ghana in 2015 Survey report in Experiment 2 gave an overall spread of the disease in the 5 selected administrative Regions in Ghana, during 2015 and delimited the incidence and severity of the disease on exotic as well as local mango varieties. However in this Experiment, the data was analyzed to reflect the percentage incidence and severity of the disease as recorded on local and exotic mango varieties in the following agro-ecological zones: Coastal savanna, Semi-deciduous forest, Transitional, and Guinea savanna zones. Results obtained are presented in Map 2, Table 3 and Appendices 3a & b. 55 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 4 Cultural, morphological and physiological characteristics of the causative organism of the mango tree decline disease in Ghana a. Isolation of the causative pathogen and studies on growth characteristics The causal agent was isolated from stringently disinfected symptomatic plant tissue of the mango plants from the survey farms. The method of isolation is described in the Materials and General Methods Section. b. Radial growth of L. theobromae on selected solid synthetic media culture under constant light (75 lux intensity) at 28±1⁰C for 72 h The synthetic agar media used were Oxytetracycline Glucose Yeast Extract Agar (OGYE), Dichloran Rose-Bengal Chloramphenicol Agar (DRBC), Czapek-Dox Agar (CDA) and Potato Dextrose Agar (PDA). The plates were inoculated at the centre with 4 mm discs of the fungus. Two lines were drawn across the centre of the plate and growth was measured along two diameters (See Materials and General Methods Section). Results obtained are summarized in Figs. 4a & 4b; Appendix 4. c. Radial growth of L. theobromae on selected solid synthetic media culture under constant darkness at 28±1⁰C for 72 h The Experiment in (b) was repeated. This time the cultures were incubated in total darkness for up to 72 h (3 days) and radial growth measured at 6 h intervals. Results are presented in Figs. 5a & 5b; Appendix 5. 56 University of Ghana http://ugspace.ug.edu.gh d. Radial growth of L. theobromae on selected solid synthetic media culture under alternating 12 h light and 12 h darkness regimes at 28±1⁰C for 72 h The constant light (75 lux intensity) and constant darkness regimes yielded almost the same result trend on all the media, with PDA and OGYE being the best. Would alternating 12 h light and 12 h darkness regime in any way influence the trend obtained in Experiments 4b and 4c? The set up was exactly the same except that the plates were moved from constant 12 h light regime into 12 h darkness in alternation for 72 h. Results obtained are presented in Figs. 6a & 6b; Appendix 6. EXPERIMENT 5 a. Radial growth of L. theobromae on different aqueous extracts of different plants parts of different origins incubated under alternating 12 h light and 12 h dark regimes at 28±1⁰C for 72 h Aqueous leaves and bark extracts of three plants, Psuedotsuga (pine needles), local mango variety, and exotic Kent mango variety were prepared in a manner described in the Materials and General Methods Section. The media were inoculated at the centre of the plates as in Experiments 4b, 4c & 4d. It was anticipated that the plant extracts will support the vegetative and radial growth of the pathogen and could serve potential hosts to the pathogen. Radial growth of colonies was measured along two diameters. Results obtained are presented in Figs. 7a & 7b; Appendix 7. 57 University of Ghana http://ugspace.ug.edu.gh b. Radial growth of L. theobromae on ethyl acetate extracts of different plants parts of different origins incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 72 h In the preceding Experiment 5a, aqueous extract pine needles (Psuedotsuga spp.) produced the fastest radial growth, followed by aqueous extract of local mango variety leaves, aqueous extract of Kent leaves, aqueous extract of local mango variety bark, aqueous extract of date palm seeds and aqueous extract of Kent variety tree bark in decreasing order. In this Experiment, it was conjectured that the polarity of the solvent may influence the yield and efficiency of the active ingredients in the plant parts. The preparation of the ethyl acetate extract, inoculation and determination of radial growth under alternating 12 h light and 12 h dark regimes as spelled out in Materials and General Methods section. Results are summarized in Figs. 8a & 8b; Appendix 8. c. Radial growth of L. theobromae on petroleum ether extracts of different plants parts of different origins incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 72 h This was a sequel to Experiment 5b but this time petroleum ether was used in the extraction of the active ingredients of Kent leaves (PKL), local mango bark (PLB), local mango leaves (PLL), Pine needles (PPN), Egyptian date palm seed (PDS), and Kent bark (PKB). The preparation of petroleum ether extract of aforementioned plant parts, inoculation of fungus and determination of radial growth under alternating 12 h light and 12 h dark regimes are spelled out in Materials and General Methods section. Results obtained are summarized in Figs. 9a & 9b; Appendix 9. 58 University of Ghana http://ugspace.ug.edu.gh d. Radial growth of L. theobromae on fruit juice agar of local mango, Kent mango and soil extract agar under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days If indeed, L. theobromae is pathogenic to the exotic Kent and the local varieties, juice extract from the fruits of these varieties should support good growth of the fungus. The pathogen is also soil borne, and the soil extract should also be able to support vegetative growth of the fungus albeit small. In this experiment, the fruit juices and the soil extracts agar were prepared in a manner described in the Materials and General Methods section. Radial growth was measured along two lines drawn at the bottom of the Petri dishes. Results are presented in Figs. 10a & 10b and Appendix 10. EXPERIMENT 6 Vegetative growth and sporulation of L. theobromae in three different media (Czapek-Dox Broth, Oxytetracycline Glucose Yeast Extract Broth, Potato Dextrose Broth) at 28±1⁰C under different light and dark regimes for 4 days There are many instances when growth of a fungus on solid agar was found to be different from when cultured in liquid media. It was conjectured that this may be attributed to differences in aeration of cultures during growth. Secondly, different light regimes may influence sporulation of fungi (Carlile et al., 2005). The objective of these experiments was to elucidate the growth differences and sporulation of the fungus in three different media namely Czapek-Dox Broth, Oxytetracycline Glucose Yeast Extract Broth, and Potato Dextrose Broth cultured in either continuous light, continuous darkness or alternating 12 h light/12 h dark regimes for 4 days. The experimental set up is described in the 59 University of Ghana http://ugspace.ug.edu.gh Materials and General Method section. Results obtained are summarized in Tables 4-6 and Appendices 11-13. EXPERIMENT 7 Vegetative growth and sporulation of L. theobromae in aqueous extract of leaves, bark and seeds of plants parts of different species incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days Results from Experiments 5 and 6 have shown that different extracts of plant parts of different plant species could support radial growth of L. theobromae on agar media. In this sequel experiment, the aqueous extract of the following plant parts were prepared as described in the Materials and General Method Section: local mango bark (ALB), local mango leaves (ALL), Kent mango bark (AKB), Kent mango leaves (AKL) bark, pine needles (APN) and Egyptian date palm seeds (ADS). Vegetative growth was assessed by the conventional dry weight method. Sporulation was assessed by mounting a piece of harvested mycelia mat under high power light microscope. Results are summarized in Table 7 and Appendix 13. EXPERIMENT 8 Vegetative growth and sporulation of L. theobromae in ethyl acetate extract of leaves, bark and seeds of plants parts of different species incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days The experimental set up was exactly as done in Experiment 7, however the extraction of the bioactive ingredients of the plant parts was carried out with ethyl acetate. Vegetative growth was assessed by the conventional dry weight method. Sporulation was assessed by mounting a piece of 60 University of Ghana http://ugspace.ug.edu.gh harvested mycelia mat under high power light microscope. Results are summarized in Table 8 and Appendix 14. EXPERIMENT 9 Vegetative growth and sporulation of L. theobromae in petroleum ether extracts of leaves, bark and seeds of plants parts of different species incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days The culture of L. theobromae grew vegetatively in all the extracts prepared with water, ethyl acetate but yield (dry weight) of the culture differed significantly. However, there was no sporulation in all instances. The petroleum ether extracts of the same plant parts were prepared with the view that growth and sporulation of the pathogen may be improved. The experimental set up was same as in Experiments 7 and 8, as described in the Materials and General Methods section. Results obtained are presented in Table 9 and Appendix 15. EXPERIMENT 10 Vegetative growth and sporulation of L. theobromae in fruit juices of Kent mango variety and local mango variety soil extract broths under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days In Experiment 5, the pathogen grew well on solid agar media containing the juice extract of local and Kent mango varieties, as well as in the soil extract agar. This may connote that these media may serve as reservoir for the pathogen in both soil and fruits. 61 University of Ghana http://ugspace.ug.edu.gh In this experiment the vegetative growth in liquid media of the above-mentioned extracts was tested. The methods used are spelled out in the Materials and General Method section, and were akin to that used in experiments 6-9. Results obtained are presented in Table 10 and Appendix 16. EXPERIMENT 11 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media under continuous light (75 lux intensity) at 28±1⁰C for 21 days Colour and colony morphology are two taxonomic characteristics used to augment other parameters in the identification of fungi. This pathogen is of no exception to the rule. Four commercially prepared synthetic and natural solid media used for laboratory identification of fungi were employed, namely Czapek-Dox Agar (CDA), Dichloran Rose-Bengal Chloramphenicol Agar (DRBC), Oxytetracycline Glucose Yeast Extract Agar (OGYE) and Potato Dextrose Agar (PDA) were assessed for their influence on colour development and change during growth of the pathogen under continuous light for 21 days. Results are shown in Plates 5-8. EXPERIMENT 12 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media under continuous darkness at 28±1⁰C for 21 days Light quality is known to influence pycnidia formation and colony characteristics of L. theobromae. In experiment 10, the plates were incubated in continuous light of 75 lux luminous intensity for 21 days. In this Experiment 12 the samples were incubated in total darkness for the same period. The Plates 9-12 show the colour and colony characteristics of the pathogen on Czapek-Dox Agar (CDA), 62 University of Ghana http://ugspace.ug.edu.gh Dichloran Rose-Bengal Chloramphenicol Agar (DRBC), Oxytetracycline Glucose Yeast Extract Agar (OGYE) and Potato Dextrose Agar (PDA) at 28±1⁰C for 21 days. EXPERIMENT 13 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media under alternating 12 h light/12 h darkness light regimes at 28±1⁰C for 21 days In this Experiment the prepared plates as described in experiments 10 and 11 were incubated at alternating 12 h light/12 h darkness light regime for the same period. Results are shown in Plates 13- 16 for cultures on CDA, DRBC, OGYE and PDA. EXPERIMENT 14 Colour change of the mycelium of L. theobromae growing on solid aqueous extract media of local mango bark, local mango leaves, Kent mango variety leaves, Kent mango variety bark, pine needles and Egyptian date palm seeds at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days The previous Experiments, 11-13, showed that light duration (continuous light, continuous darkness, alternating 12h light/12 h darkness) did not significantly influence the change of colour of mycelia colony of the fungus on different synthetic media. The aqueous extracts of the selected plant materials (local mango bark, local mango leaves, Kent mango variety leaves, Kent mango variety bark, pine needles and Egyptian date palm seeds) were used next. The preparation of the extracts, the inoculation methods and the collection of data on 63 University of Ghana http://ugspace.ug.edu.gh growth characteristics have been spelled out in the Materials and General Methods Section. Results obtained are shown in Plates 17 & 18. EXPERIMENT 15 Colour change of the mycelium of L. theobromae growing on solid ethyl acetate extract media of local mango bark, local mango leaves, Kent mango variety leaves, Kent mango variety bark, pine needles and Egyptian date palm seeds at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days This sequel experiment was a repeat of Experiment 14 but with a difference. Ethyl acetate extracts of the listed plant parts were used instead. The set up was however the same. Results obtained are presented in Plates 19 & 20. EXPERIMENT 16 Colour change of the mycelium of L. theobromae growing on solid mango fruit juice and soil extract agar at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days The fruit juice of mango should serve as a good nutrient for growth of the fungus if it is to support pathogenesis of L. theobromae. So also should the soil extracts if the pathogen can survive in soil. The experiment set up was same as in Experiments 14 and 15. Results obtained are shown in Plates 21 & 22. 64 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 17 Colony morphology of L. theobromae on different media used in Experiments 4 to 22. Colony morphology of mycelium is an important taxonomic character to augment other cultural and colour characteristics. The colony morphology of the differently formulated media in Experiments 4-22 were recorded based on the description spelt out by (Leung & Liu, 2016). The form of the culture (circular, irregular, filamentous, rhizoid), the elevation (raised, convex, flat, umbonate, crateform) and the margin of the culture (entire, undulate, filiform, curled or lobate) were recorded. Table 11 shows the results obtained. EXPERIMENT 18 Hyphal morphology of L. theobromae on different cultural formulations a. Commercial synthetic media placed under different light and dark regime The media used to assess hyphal morphology were Czapek-Dox Agar (CDA), Dichloran Rose- Bengal Chloramphenicol Agar (DRBC), Oxytetracycline Glucose Yeast Extract Agar (OGYE) and Potato Dextrose Agar (PDA). Plates 23-26 show the morphology of mycelium under the microscope of cultures placed under continuous light regime for up to 21 days at 28±1⁰C. Plates 27-30 show the morphology of mycelium under the microscope of cultures placed under continuous darkness regime for up to 21 days at 28±1⁰C. Plates 31-52 show the morphology of mycelium under the microscope of cultures placed under alternating 12 h light/12 h dark regimes for up to 21 days at 28±1⁰C. 65 University of Ghana http://ugspace.ug.edu.gh b. Aqueous extract media prepared from selected plant materials The media used to assess hyphal morphology of L. theobromae were: local mango bark (ALB), local mango leaves (ALL), Kent mango bark (AKB), Kent mango leaves (AKL), pine needles (APN), Egyptian date palm seeds (ADS). Plates 35-40 show the variation in morphology in the mycelium cultured on the various media. c. Ethyl acetate extract media prepared from selected plant materials The difference in this experiment is the solvent used in harvesting the bioactive compounds from the plant parts to support growth of the pathogen. Plates 41-46 show morphology of the mycelium cultured on different media under 12 h light/12 h darkness regimes. d. Petroleum ether extract media prepared from selected plant materials The chemical nature of the solvent for extracting the bioactive compounds from the listed plant parts may influence the morphology of the mycelium. The cultures were incubated under alternating 12 h light/12 h darkness light regimes at 28±1⁰C for 21 days. Plates 47-52 show results obtained. EXPERIMENT 19 Sporulation and conidial (spore) dimension of L. theobromae growing on differently formulated synthetic and natural media incubated at different light and dark regimes for 21 days The spore/conidia of fungi is a diagnostic parameter for authentication of identification of the species. Several factors influence spore/conidia formation including nutrients, pH, light quality, light duration and period of incubation, etc. The media used are CDA, DRBC, OGYE, PDA, local 66 University of Ghana http://ugspace.ug.edu.gh mango bark, local mango leaves, Kent mango bark, Kent mango leaves, pine needles, and Egyptian date palm seeds. The Petri plates were incubated for up to 21 days and were examined for the presence of pycnidia and number of conidia, and conidia dimension (length and width). The plates were incubated either in continuous light, continuous darkness, and alternating 12 h light and 12 h darkness. Results obtained are presented in Tables 11-14 and Plates 53 & 55. EXPERIMENT 20 Pathogenicity test of the causative pathogen on host tissue To confirm the pathogenicity of the isolated fungus to the host plant, Koch’s postulate was applied by infecting the host plant with the fungus L. theobromae. The method of inoculation and assessment of infection and severity are spelled out in the Materials and General Methods Section. Results are presented Table 15 and Plates 56 & 57. EXPERIMENT 21 Molecular characterization of the causative agent to confirm the morphology identity There have been many instances when one is dealing with a complex of species that classical microscopic and morphological features alone cannot be used to characterize an isolated pathogen since cultural morphology may vary widely on media, hosts, and environmental parameters may influence growth pattern, morphology and dimension of conidia. Sangeetha et al. (2010) showed a high degree of genetic variability within the L. theobromae isolates from banana. The identification and characterization of the isolated pathogen was done by conventional cultural and morphological characters. However in this experiment, the identity was confirmed by 67 University of Ghana http://ugspace.ug.edu.gh molecular characterization using Polymerase Chain Reactions (PCR) (See Materials and General Methods Section). The amplified products of the ITS region were purified and sequenced. The sequences of the ITS region of the 8 isolates were used in the phylogenetic studies. Details of the process of sequencing are provided in the Materials and General Methods Section. Results obtained are shown in Plate 58 (Gel electrophoregram) and Fig. 11 (Neighbour joining tree drawn with the nucleotide sequences of the ITS region of the different Lasiodiplodia species). EXPERIMENT 22 Preliminary studies on the biocontrol of L. theobromae using biotoxins from aqueous plant extracts In the concluding experiment of this thesis, the ability of plant extracts with known potent biotoxins were tried as biocontrol agents against vegetative growth of the causative agent of the mango decline syndrome in Ghana. Aqueous extracts of Chromolaena odorata leaves, Azadirachta indica leaves and Carica papaya seeds were prepared in the manner described in the Materials and General Methods section. These extracts were used in amending Potato Dextrose Broth and Potato Dextrose Agar to obtain dilutions of 1:1, 1:2, 1:5 and 1:10 v/v of the extracts. Vegetative growth in liquid culture was assessed by the oven dry weight method while radial growth was determined by measuring growth along two diameters at the bottom of the Petri plates. Results of these studies are summarized as follows: 68 University of Ghana http://ugspace.ug.edu.gh a. Radial growth of pathogen Chromelaena odorata: Figs. 12a & 12b; Appendix 19; Plate 59. Azadirachta indica: Figs. 13a & 13b; Appendix 20; Plate 60. Carica papaya: Figs. 14a & 14b; Appendix 21; Plate 61. b. Vegetative growth in liquid broth culture Chromelaena odorata: Fig. 15; Appendix 22 Azadirachta indica: Fig. 16; Appendix 23 Carica papaya: Fig. 17; Appendix 24 69 University of Ghana http://ugspace.ug.edu.gh CHAPTER 5 RESULTS EXPERIMENT 1 Field survey to assess the mango tree decline disease in the administrative regions of Ghana in 2015 a. Typical symptoms of the disease observed in the field Infected plants showed wilting of upper tips (called tip dieback) (Plate 2A); shoot dieback (Plate 2B); drying of leaves and rolling up of their margins (Plate 2C); affected leaves scorched and fell leaving a dead branch (Plate 2D). Infected branches of the plant became discoloured in the vascular tissues (Plate 3B) while the uninfected branch remained fresh and whitish (Plate 3A). As the disease advanced, exudates of gum was seen (gummosis) (Plate 2C) which became heavy as disease severity aggravated (Plate 3D). With time, the bark cracked as gummosis intensified in severe conditions (Plate 4A). The branch formed tissues beneath the stem/branch (Plate 4B). In extreme cases of pathogenesis, death of entire plant was observed (Plate 4C). The most common symptom observed was drying of leaves and rolling up of their margins (Plate 2C), followed by tip dieback (Plate 2B) and vascular discolouration (Plate 3B). 70 University of Ghana http://ugspace.ug.edu.gh b. Factors promoting expression of symptoms in the field During the survey some of the respondent farmers mentioned mechanical injury to plant through bruising and breaking by farm machinery and implements, as well as fire as being springboards for invasion of the pathogen for the mango decline syndrome. They therefore attributed these factors as those which predispose mango trees to the decline syndrome. It is well known that certain abiotic factors also contribute to the expression of the symptom of the disease under investigation. 71 University of Ghana http://ugspace.ug.edu.gh A B C D Plate 2. Typical symptoms of the mango tree decline disease observed in the field in Ghana A= Infected plant exhibiting wilting and drying of branches from the tips into the old wood (dieback) (arrowed); B=Seedling exhibiting shoot dieback (arrowed); C=Affected leaves turn brown and its margins roll upwards; D=Leaves scorch and fall, leaving a dead branch 72 University of Ghana http://ugspace.ug.edu.gh A B C D Plate 3. Typical symptoms of the mango tree decline disease observed in the field in Ghana A=Twig of healthy mango plant showing vascular tissue; B=Infected branch with discoloured vascular tissue; C= Gum appearing as a small droplet on bark of mango tree (arrowed); D=Gumming increases as disease progresses on the bark (circled) 73 University of Ghana http://ugspace.ug.edu.gh A B C Plate 4. Typical symptoms of the mango decline disease observed in the field in Ghana A= Bark cracking (with gummosis) in severe condition; B= Branch callus formation (arrowed); C= Drying of branches resulting in death of the whole tree in severe condition (arrowed) 74 University of Ghana http://ugspace.ug.edu.gh c. Knowledge of farmers on the mango tree decline disease The structured questionnaire administered to the 36 respondents showed that 84% of the respondents mistook browning of foliage as natural foliage scorching by the sun’s heat and water stress although browning of the foliage is a diagnostic symptom of the mango decline disease. Exactly 76.2% had observed abnormal cracking of the bark and vascular browning on infected trees growing on their farms. This was accompanied by oozing of latex from the uncut or undamaged portion of the trunk which 66.7% of them observed. Others (57%) recorded the abnormal drying up of twigs and branches and even eventual death of the plant. There were still 62% of them who observed as miscellany of symptoms on same trees. Eventual death of the tree was low recorded in 23.8% of the tree population. Fig. 2 shows the response of the farmers to the incidence of the typical symptoms on their mango trees. In the view of 4.8% of the respondents, excessive rainfall was presumably responsible for the mango decline disease while 14.3% of them attributed the disease to poor planting material. About 4.8% of the farmers considered poor management practices as responsible for the mango decline disease while 28.6% of farmers attributed the disease to a causal unknown pathogen. None mentioned wind and superstitious belief as responsible for the occurrence of the mango decline syndrome. Fig. 3 summarizes the results stated above. 75 University of Ghana http://ugspace.ug.edu.gh 90 80 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 Responses of farmers Fig. 2. Response (%) of 36 farmers who have observed any of the typical symptoms of the decline disease on their farms in Ghana 1=Vascular discolouration (76.2%); 2=Latex oozing from uncut portions of the trunk (66.7%); 3=Abnormal bark cracking (76.2%); 4=Abnormal drying up and death of branches (57.1%); 5=Branches drying up and breaking off (71%); 6=Stunted growth of trees (42.9%); 7=Gradual death of tress (23.8%). 76 Number of corresponds (%) University of Ghana http://ugspace.ug.edu.gh 50 40 30 20 10 0 1 2 3 4 5 6 7 8 -10 Cause of disease Fig. 3. Percentage response (%) of 36 farmers on the possible cause of the mango tree decline disease on their farms in Ghana 1=Excessive rainfall (4.8%); 2=Source of planting material (14.3%); 3=Belief and superstition (0.0%); 4=Poor soil (38.1%); 5=Poor management practices (4.8%); 6=Pathogen (28.6%); 7=Wind (0.0%); 8=Others (9.5%). 77 Number of corresponds (%) University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 2 Disease incidence and severity of mango tree decline disease in the Administrative Regions in Ghana in 2015 The survey was carried out in five out of the ten administrative regions of Ghana comprising Ashanti, Brong-Ahafo, Eastern, Greater-Accra, and Northern regions. Map 1 summarizes the percentage incidence and severity index of the disease on exotic and local mango varieties in each of the listed regions. The lowest disease incidence on the exotic Kent mango variety was 13.3% in Brong-Ahafo region, while the highest (25%) was recorded in the Ashanti region. The differences obtained were statistically significant (p≤0.05). The disease incidence on the local mango variety varied from 62.5% (Greater-Accra) to 70.0% (Ashanti region). The difference observed on the local mango variety was statistically significant (p≤0.05). Disease incidence was severer (62.5-70.0%) on the local variety of mango to 13.3-25.0% on the exotic Kent mango variety (Map 1, Table2 & Appendices 2 & 3). This data is augmented by the higher overall mean incidence for local variety (65.7%) as compared to 17.6% for the Kent exotic variety. There were no statistical different (p˃0.05) among disease incidence recorded in the Brong-Ahafo, Eastern, Greater-Accra and Northern Regions on both the exotic and local varieties of mango, albeit the highest was always recorded in Ashanti Region (Table 2, Appendices 2a & b). The disease severity index followed almost the same trend as the percentage incidence. On a scale of 0-5, the lowest severity index of 2.13 on the local variety was recorded in the Brong-Ahafo and highest recorded 2.50-2.51 in the Northern and Eastern Regions (Table 2, Map 1). The severity 78 University of Ghana http://ugspace.ug.edu.gh index was lower in the exotic variety (0.81-1.71) than the local variety (2.13-2.51) (Table 2). This was reflected in the national overall mean disease severity index which was higher in the local variety (2.38) than in the exotic variety farm (1.41) (Appendices 3a & b). 79 University of Ghana http://ugspace.ug.edu.gh Northern region Exotic Local % Incidence 15.71 66.00 Severity 1.71 2.51 index Brong-Ahafo region Exotic Local % Incidence 13.33 63.33 Severity index 1.11 2.13 Ashanti region Exotic Local % Incidence 25.00 70.00 Severity index 1.59 2.30 Eastern region Exotic Local % Incidence 16.67 66.67 Severity index 1.83 2.50 Greater Accra region Exotic Local % Incidence 17.50 62.50 Severity index 0.81 2.35 Map 1. A map of Ghana showing the disease incidence and severity in selected five administrative regions of Ghana in 2015 80 University of Ghana http://ugspace.ug.edu.gh Table 2. Mean percentage disease incidence and severity index of mango tree decline disease in 36 mango farms in five Administrative regions of Ghana in 2015 Administrative region Disease incidence (%) Mean severity index Exotic variety Local variety Exotic variety Local variety Ashanti 25.00a 70.00a 1.59b 2.39b Brong-Ahafo 13.33b 63.33b 1.11b 2.13a Eastern 16.67b 66.67b 1.83b 2.50b Greater-Accra 17.50b 62.50b 0.81a 2.35b Northern 15.71b 66.00b 1.71b 2.51b Overall mean 17.64 65.70 1.41 2.38 Means in the same column with the same alphabets are not statistically different (p≥0.05). 81 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 3 Disease incidence and severity index of the mango tree decline disease in the different agro-ecological zones of Ghana Map 2 shows the locations of the agro- ecological zones where the survey was carried out as well as the attendant % disease incidence and severity indices on either the local or the exotic mango variety plants. Clearly percentage incidence was higher in the local mango crop stands than in the exotic mango stands in all the agro- ecological zones. Furthermore, it was shown that the disease occurred in all the surveyed agro- ecological zones studied (Guinea savanna, Semi-deciduous forest; Transition, and Coastal savanna (i.e. Greater-Accra and Eastern Regions)). Table 3 which summarizes the results shows that disease incidence was >4 times more in the local variety mango crop stand than in the exotic mango crop stands in all the agro- ecological zones (Table 3). The disease incidence in the local mango variety stands varied from 60.0-74.0% as compared to 13.33-20.0% in the exotic mango crop stands. The same trend was true for the record of the severity index; it was higher in the local mango crop stand (2.28-2.70) than in the exotic crop stands (0.81-1.73). There was no statistical difference (p≥0.05) between the record of percentage disease incidence on the exotic variety in all the agro- ecological zones surveyed. The same was true for the mean severity index recorded in the local mango variety stand (Table 3; Appendices 3a & b). 82 University of Ghana http://ugspace.ug.edu.gh Table 3. Mean percentage incidence and severity index of mango tree decline disease in 36 mango farms in the indicated four agro-ecological zones of Ghana in 2015 Agro-ecological zone Disease incidence (%) Disease severity index Exotic variety Local variety Exotic variety Local variety Coastal savanna 17.50b 60.00b 0.81a 2.28b Semi-deciduous forest 15.71b 74.00a 1.71b 2.70b Transitional 20.00b 65.00b 1.73b 2.35b Guinea savanna 13.33b 65.00b 1.11b 2.39b Overall mean 16.64 66.00 1.34 2.43 Means in the same column with the same alphabets are not statistically significant (p≥0.05) 83 University of Ghana http://ugspace.ug.edu.gh Guinea savanna Exotic Local % Incidence 13.33 65.00 Severity index 1.11 2.39 Transitional Exotic Local % Incidence 20.00 65.00 Severity index 1.73 2.35 Semi-deciduous forest Exotic Local % Incidence 15.71 74.00 Severity index 1.71 2.70 Coastal savanna Exotic Local % Incidence 17.50 60.00 Severity index 0.81 2.28 Map 2. A map of Ghana showing the disease incidence and severity in four agro-ecological zones of Ghana in 2015 84 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 4 Cultural, morphological and physiological characteristics of the causative organism of the mango tree decline disease in Ghana a. Isolation of the causative pathogen A single fungal pathogen was recovered and isolated from 95% of the symptomatic mango plant tissues collected from all the farms in the four agro-ecological zones. Based on the colour, cultural and morphological characteristics on different media as spelled out in the identification manuals (Fig. 1) the fungus was tentatively identified as Lasiodiplodia theobromae, pending further physiological and molecular characterization. b. Radial growth of L. theobromae on selected solid synthetic agar media under constant light (75 lux intensity) at 28±1⁰C for 72 h Radial growth was slower on DRBC and CDA (Figs. 4a & 4b) but the superior growth on PDA approximated that of OGYE giving a mean growth of 1.67±0.27 mm/h and 1.80±0.80 mm/h respectively. On the other hand mean growth rate on CDA was 0.713±0.09 mm/h, approximating an equally slow growth rate of 0.80±0.78 mm/h on DRBC (Appendix 4). c. Radial growth of L. theobromae on selected solid synthetic agar media under constant darkness at 28±1⁰C for 72 h Under constant darkness, growth on OGYE was best in 60 h but mycelia on PDA initially lagged behind but approximated that on OGYE after 60 h. Growth of the fungus on CDA and DRBC was poorer and never approximated that of OGYE and PDA (Figs. 5a & 5b). The mean growth rate on PDA was 1.29±0.89 mm/h as compared to 1.77±0.88 mm/h on OGYE. The growth rate of 85 University of Ghana http://ugspace.ug.edu.gh mycelia on CDA was 1.17±0.76 mm/h while on DRBC it grew at the rate of 0.93±0.46 mm/h (Appendix 5). d. Radial growth of L. theobromae on selected solid synthetic media culture under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 72 h Under the alternating 12 h light and 12 h darkness, the best growth of the pathogen was on PDA and OGYE and there was no statistical difference (p˃0.05) between the growth of the fungus on OGYE and PDA. Optimum growth was attained after 54 h of incubation. On the other hand, growth of the pathogen on CDA and DRBC was slower but also close with no statistical difference (p˃0.05) (Figs. 6a & 6b). These observations were collaborated by the mean growth rates of the fungus on CDA (0.76±0.37 mm/h) OGYE (1.63±0.38 mm/h) and PDA (1.67±0.37 mm/h) (Appendix 6). Alternating 12 h light and 12 h darkness therefore better delimits the efficiency of the fungus to grow on the four media. 86 University of Ghana http://ugspace.ug.edu.gh 60 50 40 CDA 30 DRBC 20 OGYE PDA 10 0 12 18 24 Period of incubation (h) Fig. 4a. Selected hourly radial growth records of L. theobromae on the indicated synthetic media at 28±1⁰C under constant light (75 lux intensity) 100 90 80 70 60 CDA 50 DRBC 40 30 OGYE 20 PDA 10 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 4b. Radial growth of L. theobromae on indicated synthetic media at 28±1⁰C under constant light (75 lux intensity) for 72 h CDA=Czapek-Dox Agar; DRBC=Dichloran Rose-Bengal Chloramphenicol Agar; OGYE=Oxytetracycline Glucose Yeast Extract Agar; PDA=Potato Dextrose Agar. 87 Mean diameter of colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh 60 50 40 CDA 30 DRBC 20 OGYE PDA 10 0 12 18 24 -10 Period of incubation(h) Fig. 5a. Selected hourly radial growth of L. theobromae on the indicated synthetic media at 28±1⁰C under constant darkness 100 90 80 70 60 CDA 50 DRBC 40 30 OGYE 20 PDA 10 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 5b. Radial growth rate of L. theobromae on indicated synthetic media at 28±1⁰C under constant darkness for 72 h CDA=Czapek-Dox Agar; DRBC=Dichloran Rose-Bengal Chloramphenicol Agar; OGYE=Oxytetracycline Glucose Yeast Extract Agar; PDA=Potato Dextrose Agar. 88 Diameter of colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh 50 45 40 35 30 CDA 25 DRBC 20 OGYE 15 10 PDA 5 0 12 18 24 Period of incubation (h) Fig. 6a. Selected hourly radial growth of L. theobromae on indicated synthetic media at 28±1⁰C under alternating 12 h light and 12 h darkness 100 90 80 70 60 50 CDA 40 DRBC 30 OGYE 20 PDA 10 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 6b. Hourly radial growth of L. theobromae on indicated synthetic media 28±1⁰C under alternating 12h light and 12 h darkness for 72 h CDA=Czapek-Dox Agar; DRBC=Dichloran Rose-Bengal Chloramphenicol Agar; OGYE=Oxytetracycline Glucose Yeast Extract Agar; PDA=Potato Dextrose Agar. 89 Mean diameter of colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 5 a. Radial growth of L. theobromae on different aqueous extracts of plants of different origins incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 72 h L. theobromae mycelium growing on agar medium prepared from aqueous extract of pine needles (Pseudotsuga spp.) (APN) produced the fastest growth covering the entire plate in 36 h, followed by local mango leaves (ALL) and Kent exotic mango leaves (AKL) in 42 h and 48 h respectively (Figs. 7a & 7b). This is by far a superior growth of 2.14±0.38 mm/h to 2.17±0.60 mm/h as compared to ˃2.0 mm/h obtained on the commercially prepared synthetic media (Appendices 6 & 7). The remaining three aqueous extracts from local mango bark (ALB), aqueous Kent bark (AKB) and Egyptian date palm seeds (ADS) supported slower growth rate attaining a mean growth rate of 0.82±0.45 to 1.19±0.20 mm/h in 72 h (Appendix 7). b. Radial growth of L. theobromae on different ethyl acetate extracts of plants of different origins incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 72 h Mycelium of L. theobromae growing on agar medium prepared from ethyl acetate extract of pine needles (EPN), local mango leaves (ELL) and Kent mango leaves (EKL) showed the fastest growth covering the entire Petri plates in 42 h. Cultures growing on Egyptian date palm seeds (EDS) covered the plates in 60 h while the rest lagged behind (local mango bark, ELB; Kent exotic mango bark, EKB) (Figs. 8a & 8b). Growth rate of mycelium growing on agar prepared from ethyl acetate extract of pine needles (EPN) was fastest (2.48±0.26 mm/h) followed by local mango leaves, ELL (2.10±0.20 mm/h). Egyptian 90 University of Ghana http://ugspace.ug.edu.gh date palm seed, EDS (1.75±0.50 mm/h), local mango bark, ELB (1.11±0.44 mm/h) and Kent mango bark, EKB (0.75±0.58 mm/h) (Appendix 8). c. Radial growth of L. theobromae on different petroleum ether extracts of plants of different origins incubated under alternating 12 h light/12 h darkness at 28±1⁰C for 72 h The best radial growth (1.74mm/h) was obtained in petroleum ether extract of the Egyptian date palm seeds (PDS) and the least (1.20±0.06 mm/h) was obtained on local mango leaves (PLL) and local mango bark (PLB). There was however good growth on all the media. It was clear that the plant part differed in their efficiency to support growth depending on the extracting solvent (Appendix 9). d. Radial growth of L. theobromae on fruit juice agar prepared from local mango and Kent mango extract and soil extract agar incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 72 h It was conjectured that if indeed L. theobromae is pathogenic to mango and resident in soil, the extracts from mango fruits (local and exotic Kent) and soil should support good growth in vitro as well. The fungus covered the entire Petri plates containing juice from local mango and Kent (exotic) mango in 48 h (Figs. 10a & 10b) but was slow on soil extract agar attaining a mean diameter of 46.2 mm in 72 h. The fastest growth rate of the fungus (1.87 mm/h) was on local mango fruit juice followed by Kent fruit juice (1.50 mm/h) with culture of the fungus on soil extract agar trailing behind and growing at 0.62 mm/h (Appendix 10). 91 University of Ghana http://ugspace.ug.edu.gh 90 80 70 60 ALB 50 ALL 40 AKB 30 AKL 20 APN 10 ADS 0 12 18 24 Period of incubation (h) Fig. 7a. Selected hourly radial growth of L. theobromae on indicated aqueous extract of selected plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness 100 90 80 70 ALB 60 ALL 50 AKB 40 AKL 30 20 APN 10 ADS 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 7b. Radial growth of L. theobromae on aqueous extracts of selected plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 72 h ALB=Aqueous local mango bark; ALL=Aqueous local mango leaves; AKL=Aqueous Kent leaves; APN=Aqueous pine needles; ADS= Aqueous Egyptian date palm seeds 92 Mean diameter of colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh 70 60 50 ELB 40 ELL 30 EKB EKL 20 EPN 10 EDS 0 12 18 24 Period of incubation (h) Fig. 8a. Radial growth of L. theobromae on ethyl acetate extracts of selected plant materials at 28±1⁰C under alternating 12h light and 12 h darkness 100 90 80 70 ELB 60 ELL 50 EKB 40 EKL 30 EPN 20 10 EDS 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 8b. Radial growth rate of L. theobromae on ethyl acetate extracts of selected plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 72 h ELB=Ethyl acetate local mango bark; ELL=Ethyl acetate local mango leaves; EKL=Ethyl acetate Kent leaves; EPN=Ethyl acetate pine needles; EDS=Ethyl acetate Egyptian date palm seeds 93 Mean diameter of colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh 60 50 40 PLB PLL 30 PKB 20 PKL PPN 10 PDS 0 12 18 24 Period of incubation (h) Fig. 9a. Selected hourly radial growth of L. theobromae on petroleum ether extracts of selected plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness 100 90 80 70 PLB 60 PLL 50 PKB 40 PKL 30 20 PPN 10 PDS 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 9b. Radial growth rate of L. theobromae on the indicated petroleum ether extracts of selected plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 72 h PLB=Petroleum ether local mango bark; PLL=Petroleum ether local mango leaves; PKL=Petroleum ether Kent leaves; PPN= Petroleum ether pine needles 94 Mean diameter of colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh 60 50 40 30 JLF JKF 20 SAE 10 0 12 18 24 Period of incubation (h) Fig. 10a. Selected hourly radial growth of L. theobromae on mango fruit juice and soil extracts at 28±1⁰C under alternating 12 h light and 12 h darkness 100 90 80 70 60 50 JLF 40 JKF 30 SAE 20 10 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 10b. Radial growth of L. theobromae on mango fruit juice and soil extracts at 28±1⁰C under alternating12 h light and 12 h darkness for 72 h JLF=Juice from local mango fruits; JKF=Juice from Kent (exotic) mango fruits; SEA=Soil extract agar 95 Mean diameter of colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 6 Vegetative growth of L. theobromae in three different commercially prepared media under different light/dark regimes at 28±1⁰C for 4 days a. Growth under continuous light The fungus grew well in all the media but to a different extent. The best growth was on Czapek-Dox Broth (CDB) followed by OGYE broth and Potato Dextrose Broth (PDB) in decreasing order (Table 4; Appendix11) the differences observed were statistically significant (p≤0.05). The pH drifted from neutral to acid side in CDB and from neutral to basic side in OGYE-B, which in PDB, there was only a slight shift to the acidic side (Table 4). b. Growth under continuous darkness L. theobromae accumulated dry matter just as under continuous light but grew best again in CDB (427.5±2.5 mg) followed by OGYE-Broth (290.0±10.8 mg) and PDB (147.5±9.47 mg) in decreasing order. The differences observed were statistically significant (p≤0.05) (Table 5; Appendix 12) the drift in pH was similar to what was recorded under continuous light. c. Growth under alternating 12 h light/12 h darkness The trend was the same as in the culture incubated in continuous light, and continuous darkness. The best growth of the fungus was in CDB (430.0±4.08 mg) followed by OGYE-B (335.0±2.89 mg) and PDB (137.5±2.50 mg) in decreasing order. The quantity and duration of light or darkness did not seem to have influenced vegetative growth of the fungus. The type of media however significantly (p≤0.05) influence dry matter accumulation as the drift in pH was similar under all the different light 96 University of Ghana http://ugspace.ug.edu.gh regimes employed (Table 6; Appendix 13). Interestingly, there was no sporulation in all the cultures irrespective of the light regime used. This is presumably because of the short incubation period. 97 University of Ghana http://ugspace.ug.edu.gh Table 4. Vegetative growth and sporulation of L. theobromae in indicated liquid media at 28±1⁰C under continuous light for 4 days Number of Type pH of media Dry weight Mean dry spores per 20- of of wt. of Rep 25 microscope media mycelium mycelium Initial Final field views (mg) ±S.E(mg) 1 5.08 440.0* CDB 2 7.14 5.10 440.0 432.5±4.79a - 3 5.07 420.0 1 8.22 290.0 OGYE-B 2 7.10 9.00 310.0 290.0±7.07b - 3 9.2 280.0 1 4.10 130.0 PDB 2 4.23 4.16 140.0 140.0±4.08c - 3 4.05 150.0 Rep=Replicate; (±)SE=Standard error; CDB=Czapek-Dox Broth; OGYE-B=Oxytetracycline Glucose Yeast Extract Broth; PDB=Potato Dextrose Broth; -= Nil. Means in the same column followed by different alphabets are significantly different at p≤0.05. * Data corrected to the nearest whole number 98 University of Ghana http://ugspace.ug.edu.gh Table 5. Vegetative growth and sporulation of L. theobromae in indicated liquid media at 28±1⁰C under continuous darkness for 4 days Number of Type pH of media Dry weight Mean dry spores per 20- of of wt. of Rep 25 microscope media Mycelium mycelium Initial Final field views (mg) ±S.E(mg) 1 5.10 430.0* CDB 2 7.14 5.09 430.0 427.5±2.5a - 3 5.18 420.0 1 9.00 320.0 OGYE-B 2 7.10 9.02 290.0 290.0±10.8b - 3 8.53 280.0 1 4.20 150.0 PDB 2 4.23 4.05 160.0 147.5±9.47 c - 3 4.10 120.0 Rep=Replicate; (±)=Standard error; CDB=Czapek-Dox Broth; OGYE-B=Oxytetracycline Glucose Yeast Extract Broth; PDB=Potato Dextrose Broth; - = Nil. Means in the same column followed by different alphabets are significantly different at p≤0.05. * Data corrected to the nearest whole number 99 University of Ghana http://ugspace.ug.edu.gh Table 6. Vegetative growth and sporulation of L. theobromae in indicated liquid media at 28±1⁰C under alternating 12 h light and 12 h darkness for 4 days Dry weight Number of Type Mean dry pH of media of spores per 20- of wt. of Rep mycelium 25 microscope media mycelium Initial Final (mg) field views ±S.E(mg) 1 5.10 430.0* CDB 2 7.14 5.10 440.0 430.0±4.08a - 3 5.20 430.0 1 8.20 330.0 OGYE-B 2 7.10 8.30 340.0 335.0±2.89b - 3 8.20 330.0 1 4.40 140.0 PDB 2 4.23 4.40 140.0 137.5±2.50c - 3 4.30 140.0 Rep=Replicate; (±)SE=Standard error; CDB=Czapek-Dox Broth; OGYE-B=Oxytetracycline Glucose Yeast Extract Broth; PDB=Potato Dextrose Broth; -=Nil. Means in the same column followed by different alphabets are significantly different at p≤0.05. * Data corrected to the nearest whole number 100 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 7 Vegetative growth and sporulation of L. theobromae in aqueous extracts of leaves, bark and seeds of plant parts from different species incubated under 12 h light/12 h dark regimes at 28±1⁰C for 4 days The aqueous extracts of selected plant parts (local mango leaves, ALL; Kent (exotic) mango leaves, AKL; local mango bark, ALB; Kent (exotic) mango bark, AKB; Pine needles, APN; and Egyptian date palm seeds, ADS) supported luxuriant growth of the pathogen in liquid culture ranging from 225.5±2.50 mg (Kent exotic bark, AKB) to 561.7±1.44 mg (local mango bark, ALB) (Table 7; Appendix 14). The pH of the media drifted either to the more acid side or basic side. The plant parts tested therefore have the potential to serve as nutrient for L. theobromae. In all instances, no sporulation was detected in the media owing to the short incubation period. 101 University of Ghana http://ugspace.ug.edu.gh Table 7. Vegetative growth and sporulation of L. theobromae in aqueous extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness Number of Type Dry weight Mean dry Rep spores per 20- of pH of media of wt. of 25 media Mycelium mycelium microscope (mg) ±S.E(mg) Initial Final field views 1 6.68 560.0* ALB 2 5.80 6.73 560.0 561.7±1.44a - 3 6.70 565.0 1 5.10 530.0 ALL 2 4.70 5.00 540.0 542.5±6.29b - 3 5.20 560.0 1 6.60 250.0 AKB 2 7.81 6.50 260.0 225.5±2.50c - 3 6.50 255.0 1 5.30 370.0 AKL 2 4.90 5.40 380.0 377.5±4.79d - 3 5.20 390.0 1 5.70 410.0 APN 2 5.00 5.80 390.0 402.5±4.79e - 3 5.40 400.0 1 5.60 530.0 ADS 2 6.23 5.32 540.0 538.3±3.82b - 3 5.62 545.0 Rep=Replicate; (±)=Standard error; ALB=Aqueous local mango bark; ALL=Aqueous local mango leaves; AKB=Aqueous Kent bark; AKL=Aqueous Kent mango leaves; APN=Aqueous pine needles; ADS=Aqueous Egyptian date palm seeds; - =Nil Means in the same column followed by different alphabets are significantly different at p≤0.05. * Data corrected to the nearest whole number 102 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 8 Vegetative growth and sporulation of L. theobromae in ethyl acetate extracts of leaves, bark and seeds of plant parts from different species incubated under 12 h light/12 h darkness regimes at 28±1⁰C for 4 days It was presumed that the nature of solvent used in the extraction of the active ingredients on the plant parts may influence the growth of the pathogen in vitro. Interestingly, all the media used supported good dry matter accumulation ranging from 336.7±2.89 mg (local mango bark) to 222.5±2.50 mg (Kent exotic mango leaves). However this range of dry weight was inferior to the range of 225.5±2.50 – 561.7±1.44 mg obtained in the results of the aqueous extracts. Table 8 and Appendix 15 show the results obtained. In all instances none of the mycelium in the Petri plates produced pycnidia or conidia. 103 University of Ghana http://ugspace.ug.edu.gh Table 8. Vegetative growth and sporulation of L. theobromae in ethyl acetate extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and darkness Number of Type Dry weight Mean dry Rep spores per 20- of pH of media of wt. of 25 media mycelium mycelium microscope (mg) ±S.E(mg) Initial Final field views 1 5.58 330.0* ELB 2 4.70 5.73 340.0 a336.7±2.89 - 3 5.50 340.0 1 5.90 230.0 ELL 2 4.80 b6.20 220.0 223.3±2.89 - 3 6.10 220.0 1 7.00 320.0 EKB 2 7.88 6.80 320.0 c316.7±2.89 - 3 6.82 310.0 1 5.60 220.0 EKL 2 5.30 5.60 230.0 b 222.5±2.50 - 3 5.60 220.0 1 5.90 310.0 EPN 2 6.05 5.80 320.0 c313.3±2.89 - 3 5.7 310.0 1 5.28 260.0 EDS 2 6.60 5.20 260.0 f263.3±2.89 - 3 5.10 270.0 Rep=Replicate; SE(±)=Standard error; ELB=Ethyl acetate local mango bark; ELL=Ethyl acetate local mango leaves; EKL=Ethyl acetate Kent leaves; EKB=Ethyl acetate Kent bark; EPN=Ethyl acetate pine needles; EDS=Ethyl acetate Egyptian date palm seeds; - =Nil. Means in the same column followed by different alphabets are significantly different at p≤0.05. * Data corrected to the nearest whole number 104 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 9 Vegetative growth and sporulation of L. theobromae in petroleum ether extracts of leaves, bark and seeds of plant parts from different species incubated under alternating 12 h light/12 h dark regimes at 28±1⁰C for 4 days The culture of L. theobromae grew vegetatively in all the extract prepared with water and ethyl acetate but yield (dry weight) of the culture differed significantly and there was no sporulation of mycelium growing in the presence of the water and ethyl acetate. The petroleum ether extract also supported good vegetative growth (Table 9). The best growth was recorded in Kent mango bark, PKB (358.3±1.44 mg) followed by local mango leaves, PLL (325.0±4.3 mg); the least growth was obtained in mycelium culture of petroleum ether extract of local mango bark, PLB (216.7±2.89 mg). There was no sporulation by all the cultures presumably because of the short incubation period of 4 days. It was however clear that despite the variation in polarity of the solvents used in extracting the bioactive compounds from the plat parts, appreciable vegetative growth of the pathogen was recorded (Table 9; Appendix 16) but no sporulation was recorded. 105 University of Ghana http://ugspace.ug.edu.gh Table 9. Vegetative growth and sporulation of L. theobromae in petroleum ether extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and darkness Type Dry weight Mean dry Sporulation Rep of pH of media of wt. of per 20-25 media mycelium mycelium microscope (mg) ±S.E(mg) field views Initial Final 1 5.90 220.0* PLB 2 4.90 5.20 210.0 216.7±2.89a - 3 5.10 220.0 1 5.58 320.0 PLL 2 5.70 5.70 320.0 325.0±4.3b - 3 5.50 335.0 1 6.89 360.0 PKB 2 7.18 6.80 360.0 358.3±1.44c - 3 6.82 355.0 1 5.54 220.0 PKL 2 5.55 5.50 220.0 223.3±2.89 a - 3 5.50 230.0 1 6.00 310.0 PPN 2 6.15 5.90 315.0 311.7±1.44b - 3 5.80 310.0 1 6.45 240.0 PDS 2 6.65 6.30 245.0 238.3±3.82d - 3 6.40 230.0 Rep=Replicate; SE(±)=Standard error; PLB=Petroleum ether local mango bark; PLL=Petroleum ether local mango leaves; PKB=Petroleum ether Kent bark; PKL=Petroleum ether Kent mango leaves; PPN=Petroleum ether pine needles; PDS=Petroleum ether Egyptian date palm seeds; - =Nil. Means in the same column followed by different alphabets are significantly different at p≤0.05. * Data corrected to the nearest whole number 106 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 10 Vegetative growth of L. theobromae in fruit juices of Kent mango variety and local mango variety and soil extract broths under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 4 days In Experiment 5, the pathogen grew well radially on solid agar media containing juice extracts of local and exotic Kent mango varieties as well as Soil Extract Agar. This was attributed to the fact the nutrients in the host may be supportive of the growth of L. theobromae. The dry matter accumulation by L. theobromae in liquid culture was best in local mango fruit extract, JLF (598.3±3.81 mg) followed by the juice extract of Kent mango variety. Growth in the Soil Extract Agar was about one-third (1/3) to one-quarter (1/4) what obtained in the fruit juice extract (Table 10; Appendix 17). Again, there was no sporulation owing to the short incubation time of 4 days. 107 University of Ghana http://ugspace.ug.edu.gh Table 10. Vegetative growth and sporulation of L. theobromae in mango fruit juice and soil extracts media at 28±1⁰C under alternating 12 h light/12 h darkness regimes Type Rep Mean dry Sporulation of pH of media Dry weight of wt. of per 20-25 media Mycelium (mg) mycelium microscope ±S.E(mg) field views Initial Final 1 5.60 590.0* JLF 2 5.33 5.23 600.0 598.3±3.81a - 3 5.60 605.0 1 5.50 410.0 JKF 2 5.50 5.60 420.0 416.7±2.89 b - 3 5.40 420.0 1 6.31 150.0 SEA 2 5.30 6.06 160.0 153.3±2.89c - 3 6.05 150.0 Rep=Replicate; ±SE=Standard error; JLF=Juice from local mango fruits; JKF=Juice from exotic (Kent) mango fruits; SEA=Soil extract agar; - = Nil. Means in the same column followed by different alphabets are significantly different at p≤0.05. * Data corrected to the nearest whole number 108 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 11 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media incubated under continuous light (75 lux intensity) at 28±1⁰C for 21 days Colour changes in the mycelium growing in continuous light on different agar media are shown in Plate 5 (Czapek-Dox Agar); Plate 6 (Dichloran Rose-Bengal Chloramphenicol Agar); Plate 7 (Oxytetracycline glucose yeast agar); Plate 8 (Potato Dextrose Agar). The pathogen (L. theobromae) in continuous light initially has shades of ochre to white in the actively growing state, turns fluffy white to grey. The whitish or greyish mycelium steadily becomes pigmented into black after 8-12 days, deepening in intensity of black depending on the media (Plates 5-8). 109 University of Ghana http://ugspace.ug.edu.gh A B C D Plate 5. Colour change of colony of L. theobromae on Czapek-Dox Agar(CDA) at 28±1⁰C under continuous light Growth period: A=2 days; B=7 days;C=14 days; D=21 days (Note the change in colour from whitish to grey and finally black) A B C D Plate 6. Colour change of colony of L. theobromae on Dichloran Rose-Bengal Chloramphenicol Agar (DRBC)at 28±1⁰C under continuous light Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from white to greyish) . 110 University of Ghana http://ugspace.ug.edu.gh A B C D Plate 7. Colour change of colony of L. theobromae on Oxytetracycline Glucose Yeast Agar (OGYE) at 28±1⁰C under continuous light Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from whitish to greyish black) A B C D Plate 8. Colour change of colony of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under continuous light Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from whitish to grey and finally black) 111 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 12 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media incubated under continuous darkness at 28±1⁰C for 21 days Colour changes in the mycelium of the test fungus growing on different agar media (CDA, DRBC, OGYE and PDA) growing in total continuous darkness are shown in Plate 9 (CDA), Plate 10 (DRBC), Plate 11 (OGYE) and Plate 12 (PDA). The colour change was essentially the same on all the selected synthetic media as was observed under continuous light. The whitish mycelium gradually changed to grey and finally black after 7- 12 days incubation period. Colour changed from the centre of the Petri plates and radiated to the edges of the Petri dishes. The pigmentation was therefore not affected by the duration of light or darkness. . 112 University of Ghana http://ugspace.ug.edu.gh A B C D Plate 9. Colour change of colony of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under continuous darkness Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from whitish to grey and finally black). A B C D ggg gg Plate 10. Colour change of colony of L. theobromae on Dichloran Rose-Bengal Chloramphenicol Agar (DRBC) at 28±1⁰C under continuous darkness Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from whitish to grey and finally black) 113 University of Ghana http://ugspace.ug.edu.gh A B C D Plate 11. Colour change of colony of L. theobromae on Oxytetracycline Glucose Yeast Agar (OGYE) at 28±1⁰C under continuous darkness Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from white to greyish and finally black) A B C D Plate 12. Colour change of colony of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under continuous darkness Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from whitish to grey and finally black) 114 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 13 Colour change of mycelium of L. theobromae growing on four commercially prepared synthetic solid media incubated under alternating 12 h light/12 h darkness regimes at 28±1⁰C for 21 days Changes in colour of the mycelium of L. theobromae placed under alternating 12 h light and12 h darkness regimes at 28±1⁰C for 21 days are shown in Plate 13 (CDA), Plate 14 (DRBC), Plate 15 (OGYE) and Plate 16 (PDA). As in the case of the samples kept in continuous light and continuous darkness, changes in colour of the mycelium of the test fungus was essentially the same on all the media and was not significantly influenced by different light regimes. 115 University of Ghana http://ugspace.ug.edu.gh A B C D GG Plate 13. Colour change of colony of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from whitish to greyish black) A B C D Plate 14. Colour change of colony of L. theobromae on Dichloran Rose-Bengal Chloramphenicol Agar (DRBC) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from white to grey and finally black) 116 University of Ghana http://ugspace.ug.edu.gh A B C D Plate 15. Colour change of colony of L. theobromae on Oxytetracycline Glucose Yeast Agar (OGYE) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from white to greyish black) A B C D Plate 16. Colour change of colony of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under 12 h alternating light and 12 h darkness Growth period: A=2 days; B=7 days; C=14 days; D=21 days (Note the change in colour from whitish to grey and finally black) 117 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 14 Colour change of the mycelium of L. theobromae growing on solid aqueous extract media of local mango bark, local mango leaves, Kent mango variety leaves, Kent mango variety bark, pine needles and Egyptian date palm seeds at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days The 12 h light/12 h darkness regimes was chosen for the subsequent studies as colouration of mycelium under the different light regimes was found to be the same. The mycelium, irrespective of the medium used changed from white to grey to black. Plates 17-18 show colour change of the mycelium growing in aqueous extracts of local mango bark (Plate 18A), local mango leaves (Plate 18B), Kent mango bark (18C), Kent mango leaves (Plate 18D), Pine needles (Plate 18E) and Egyptian date palm seeds (Plate 18F) after 7 days and 14 days incubation. 118 University of Ghana http://ugspace.ug.edu.gh A B C D E F Plate 17. Colour change of colony of L. theobromae on aqueous extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 5 days A=local mango bark; B= local mango leaves; C= Kent mango leaves; D= Kent mango bark; E= pine needles; F=Egyptian date palm seeds. 119 University of Ghana http://ugspace.ug.edu.gh A B C D E F Plate 18. Colour change of colony of L. theobromae on aqueous extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 darkness for 14 days A=local mango bark; B=local mango leaves; C=Kent leaves; D=Kent mango bark; E=pine needles; F=Egyptian date palm seeds 120 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 15 Colour change of the mycelium of L. theobromae growing on solid ethyl acetate extract media of local mango bark, local mango leaves, Kent mango variety leaves, Kent mango variety bark, pine needles and Egyptian date palm seeds at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days In all instances the white mycelium steadily changed from fluffy white to greyish black after 10-15 days of incubation (Plate 20). Plates 19 and 20 show the change in colour of the mycelium from white to greyish black in 15 days in all the test samples. 121 University of Ghana http://ugspace.ug.edu.gh A B C D V V V E E E E E E E F Plate 19. Colour change of colony of L. theobromae on ethyl acetate extracts of indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 5 days A=local mango bark; B=local mango leaves; C=Kent mango leaves; D=Kent mango bark; E=pine needles; F=Egyptian date palm seeds. 122 University of Ghana http://ugspace.ug.edu.gh A B C D V VE E E E E E E F DD V V Plate 20. Colour change of colony of L. theobromae on ethyl acetate extracts of indicated plant E E E E materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 14 days A=local mango bark; B=local mango leaves; C=Kent mango leaves; D=Kent mango bark; E=pine needles; F=Egyptian date palm seeds. 123 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 16 Colour change of the mycelium of L. theobromae growing on solid mango fruit juice and soil extract agar at 28±1⁰C under alternating 12 h light/12 h darkness light regimes for 21 days The mango fruit juice is a potential host of the pathogen and the soil could harbour a reservoir of the pathogen in the field. Plates 21 and 22 show the results obtained. The mycelium was whitish and gradually turned grey intensity in colour to black after 21 days. The culture on soil extract agar changed steadily from grey, turned black from the central portion of the colony and extending eventually to the edge of the Petri plate (Plates 21 & 22). 124 University of Ghana http://ugspace.ug.edu.gh A B V V E E E E Plate 21. Colour change of colony of L. theobromae on indicated natural media at 28±1⁰C under alternating 12 h light and 12 h darkness for 5 days A=Juice from mango fruits; B=Soil extract agar A B V V E E E E Plate 22. Colour change of colony of L. theobromae on indicated natural media at 28±1⁰C under alternating 12 h light and 12 h darkness for 14 days A=Juice from mango fruits; B=Soil extract agar 125 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 17 Colony morphology of L. theobromae on different mycological media used in these Experiments Table 11 summarizes the morphology of the culture colony of L. theobromae. It shows the Form, Elevation and Margin morphology as described by (Leung & Liu, 2016) (see Materials and General Methods). The Form of the colonies were generally all Circular; the Elevation was either Flat, Raised or Umbulate. The margin were generally Undulate, Entire or Filliform. The morphology of the colonies were not significantly influenced by the three different light regimes namely, continuous light, continuous darkness, and alternating 12 h light/12 h darkness (Table 11). 126 University of Ghana http://ugspace.ug.edu.gh Table 11. Morphology of the colonies of L. theobromae culture on the indicated media at 28±1⁰C exposed to three different light regimes Colony morphology Medium of growth Form Elevation Margin CDA Circular Flat Undulated DRBC Circular Raised Entire OGYE Circular Flat Filiform PDA Circular Flat Filiform ALL Circular Flat Entire ALB Circular Flat Entire AKL Circular Flat Entire AKB Circular Flat Entire ADA Circular Flat Entire APN Circular Flat Entire ELL Circular Flat Entire ELB Circular Flat Entire EKL Circular Flat Entire EKB Circular Flat Entire EDA Circular Flat Entire EPN Circular Flat Entire PLL Circular Flat Entire PLB Circular Flat Filiform PKL Circular Flat Filiform PKB Circular Flat Filiform PDA Circular Flat Filiform PPN Circular Flat Filiform SEA Circular Flat Filiform JLF Circular Flat Entire JLF Circular Flat Entire CDA=Czapek-Dox Agar; DRBC=Dichloran Rose-Bengal Chloramphenicol Agar; OGYE=Oxytetracycline Glucose Yeast Extract Agar; PDA=Potato Dextrose Agar; ALB=Aqueous local mango bark; ALL=Aqueous local mango leaves; AKL=Aqueous Kent mango leaves; APN=Aqueous pine needles; ADS= Aqueous Egyptian date palm seeds; ELB=Ethyl acetate local mango bark; ELL=Ethyl acetate local mango leaves; EKL=Ethyl acetate Kent mango leaves; EPN=Ethyl acetate pine needles; EDS=Ethyl acetate Egyptian date palm seeds; PLB=Petroleum ether local mango bark; PLL=Petroleum ether local mango leaves; PKB=Petroleum ether Kent mango bark; PKL=Petroleum ether Kent mango leaves; PPN= Petroleum ether pine needles, PDS=Petroleum ether Egyptian date palm seeds; JLF=Juice from local mango fruits; JKF=Juice from Kent (exotic) mango fruits; SEA=Soil extract agar 127 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 18 Hyphal morphology of L. theobromae under different cultural formulations a. Hyphal morphology on commercially synthesized media (CDA, DRBC, OGYE, PDA) incubated at different light/darkness regimes for 21 days at 28±1⁰C The young mycelium of the fungus on all the test media (CDA, DRBC, OGYE, PDA) growing under continuous light conditions were similar when viewed under the high power (x100) of the light microscope. They were initially hyaline and non-septate with a diameter of ≤2.0±0.5µm. The diameter of the mycelium increased with age from about ≤2.0±0.5 µm in 2 days to about ≤8.0±1.2 µm in 21 days (Plates 23-26, continuous light); (Plates 27-30, continuous darkness); (Plates 31-34, alternating 12 h light/12 h darkness). It was observed that the morphology of the mycelium (hyphae) was not influenced by the different light regimes (continuous light, continuous dark, and continuous dark). The formation of septa commenced after 2 days and as the incubation period was prolonged, “chlamydospores-like” structures where formed by the hyphae (Plates 23-34) especially on PDA, CDA and DRBC. b. Hyphal morphology of L. theobromae culture in different aqueous extracts of different plant parts incubated under alternating12 h light/12 h darkness for 21 days at 28±1⁰C The alternating 12 h light and 12 h dark regimes was chosen for the experiment. The mycelium viewed under x100 and x400 magnifications were initially (2 days) hyaline, non-septate with diameter of ≤2.0±1.3 µm. Septation commenced after 2 days with an increase in diameter of mycelium up to 2.0-8.0±1.7 µm (Plates 35-39) in 21 days. Darkening of mycelium and 128 University of Ghana http://ugspace.ug.edu.gh “chlamydospore-like” formation set in after 7 days. In some instances, rounding off to form chlamydospore-like structures (Plates 41C & 42C) was accompanied by the thickening of the wall of the hyphae. This was not observed in the commercially synthesized media (CDA, DRBC, OGYE, PDA). On the other hand the mycelium of the fungus formed rounded chlamydospore-like structures in pine needle extract (Plate 39C). c. Hyphal morphology of L. theobromae culture in different ethyl acetate extracts of different plant parts incubated under alternating12 h light/12 h darkness for 21 days at 28±1⁰C The mycelium of L. theobromae viewed under x100 and x400 magnifications of light microscope were initially hyaline after 2 days and remained non-septate with a diameter (≤2.0±1.3 µm) similar to the aqueous extracts of the same plant parts. Septation commenced after 2 days with an increase in diameter of mycelium to 2.0-15.0±1.5 µm (Plates 41-46). Rounding off of mycelium to form “chlamydospore-like” structures after 21 days was observed on local mango leaves (Plate 43C) and pine needles (Plate 45C). In the case of the local mango bark, large “turbinate-like” cells with wall thickening was formed (Plate 41C) as well, while the mycelium of L. theobromae growing on Kent leaves extract was vacuolated and the cell wall was thickened (Plate 44C). d. Hyphal morphology of L. theobromae culture in different petroleum ether extracts of different plant parts incubated under alternating12 h light/12 h darkness for 21 days at 28±1⁰C The development of the hyphae viewed under low (x40) and high power (x400) magnifications were initially hyaline and remained non-septate till after 2 days when septate were formed. The features of the resulting mycelium was akin to what existed in the aqueous and ethyl 129 University of Ghana http://ugspace.ug.edu.gh acetate extract. However L. theobromae growing on Kent mango bark was vacuolated and the cell walls thickened (Plate 49C) similar to what was observed in L. theobromae growing in ethyl acetate Kent mango leaves extract (Plate 44C). 130 University of Ghana http://ugspace.ug.edu.gh 5µm A 12µm B 12µm C Plate 23. Photograph showing the hyphae of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under continuous light for 21 days Growth period: A=2 days; B=7 days; C=21days 8µm A 10µm B 8µm C Plate 24. Photograph showing the hyphae of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under continuous light for 21 days Growth period: A=2 days; B=7 days; C=21days 131 University of Ghana http://ugspace.ug.edu.gh 5µm A 6µm B 10µm C Plate 25. Photograph showing the hyphae of L. theobromae on Dichloran Rose-Bengal Chloramphenicol agar (DRBC) at 28±1⁰C under continuous light for 21 days Growth period: A=2 days; B=7 days; C=21days 5µm A 6µm B 10µm C Plate 26. Photograph showing the hyphae of L. theobromae on Oxytetracycline Glucose Yeast Extract agar (OGYE) at 28±1⁰C under continuous light for 21 days Growth period: A=2days; B=7days; C=21days 132 University of Ghana http://ugspace.ug.edu.gh 7µm A 7µm B 15µm C A Plate 27. Photograph showing the hyphae of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under continuous darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 5µm A 7µm B 10µm C Plate 28. Photograph showing the hyphae of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under continuous darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 133 University of Ghana http://ugspace.ug.edu.gh 4µm A 7µm B 8µm C 7µm Plate 29. Photograph showing the hyphae of L. theobromma e on Dichloran Rose-Bengal Chloramphenicol Agar (DRBC) at 28±1⁰C under continuous darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 4µm A 8µm B 7µm C Plate 30. Photograph showing the hyphae of L. theobromae on Oxytetracycline Glucose Yeast Extract Agar (OGYE) at 28±1⁰C under continuous darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 134 University of Ghana http://ugspace.ug.edu.gh 5µm A 8µm B 12µm C B Plate 31. Photograph showing the hyphae of L. theobromae on Potato Dextrose Agar (PDA) at 28±1⁰C under alternating 12h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 5µm A 10µm B 10µm C Plate 32. Photograph showing the hyphae of L. theobromae on Czapek-Dox Agar (CDA) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 135 University of Ghana http://ugspace.ug.edu.gh 5µm A 7µm B 10µm C Plate 33. Photograph showing the hyphae of L. theobromae on Dichloran Rose-Bengal Extract agar (DRBC) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 5µm A 8µm B 8µm C Plate 34. Photograph showing the hyphae of L. theobromae on Oxytetracycline Glucose Yeast Extract Agar (OGYE) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 136 University of Ghana http://ugspace.ug.edu.gh 2µm A 6µm B 9µm C Plate 35. Photograph showing the hyphae of L. theobromae on aqueous extract of local mango bark (ALB) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 1µm A 7µm B 8µm C Plate 36. Photograph showing the hyphae of L. theobromae on aqueous extract of local mango leaves (ALL) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 137 University of Ghana http://ugspace.ug.edu.gh 2µm A 10µm B 10µm C A A Plate 37. Photograph showing the hyphae of L. theobromae on aqueous extract of Kent mango bark (AKB) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 2µm A 10µm B 12µm C A A A Plate 38. Photograph showing the hyphae of L. theobromae on aqueous extract Kent mango leaves (AKL) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 138 University of Ghana http://ugspace.ug.edu.gh 2µm A 7µm B 12µm C A Plate 39. Photograph showing the hyphae of L. theobromae on aqueous extract of pine needles (APN) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 2µm A 7µm B 12µm C A Plate 40. Photograph showing the hyphae of L. theobromae on aqueous extract of Egyptian date palm seeds (ADS) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 139 University of Ghana http://ugspace.ug.edu.gh 2µm A 8µm B 15µm C A A A Plate 41. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of local mango bark (ELB) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 2µm A 6µm B 12µm C A A A Plate 42. Photograph showing the hyphae of L. theobromae on ethyl acetate extracts of local mango leaves (ELL) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 140 University of Ghana http://ugspace.ug.edu.gh 2µm A 10µm B 8µm C A A A Plate 43. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of Kent mango bark (EKB) at 28±1⁰C under alternating 12 h light and 12 h darkness Growth period: A=2 days; B=7 days; C=21days 2µm A 10µm B 8µm C A A A Plate 44. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of Kent mango leaves (EKL) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 141 University of Ghana http://ugspace.ug.edu.gh 2µm A 7µm B 8µm C A A A Plate 45. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of pine needles (EPN) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 2µm A 7µm B 8µm C A A A Plate 46. Photograph showing the hyphae of L. theobromae on ethyl acetate extract of Egyptian date palm seeds (EDS) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 142 University of Ghana http://ugspace.ug.edu.gh 5µm A 7µm B 10µm C A A A Plate 47. Photograph showing the hyphae of L. theobromae on petroleum ether extract of local mango bark (PLB) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 5µm A 7µm B 10µm C A A A Plate 48. Photograph showing the hyphae of L. theobromae on petroleum ether extract of local mango leaves (PKB) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 143 University of Ghana http://ugspace.ug.edu.gh 5µm A 7µm B 10µm C A A A Plate 49. Photograph showing the hyphae of L. theobromae on petroleum ether extract of Kent mango bark (PKB) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 5µm A 6µm B 10µm C A A A Plate 50. Photograph showing the hyphae of L. theobromae on petroleum ether extract of Kent mango leaves (PKB) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 144 University of Ghana http://ugspace.ug.edu.gh 2µm A 7µm B 10µm C A A A Plate 51. Photograph showing the hyphae of L. theobromae on petroleum ether extract of pine needles (PPN) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 3µm A 7µm B 10µm C A A A Plate 52. Photograph showing the hyphae of L. theobromae on petroleum ether extract of date palm seeds (PDS) at 28±1⁰C under alternating 12 h light and 12 h darkness for 21 days Growth period: A=2 days; B=7 days; C=21days 145 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 19 Sporulation in culture and conidial dimension of L. theobromae growing on differently formulated synthetic and natural media incubated at different light/dark regimes at 28±1⁰C for 25 days None of the plates containing CDB, OGYE, DRBC, PDA, and neither aqueous, ethyl acetate and petroleum ether extracts of local mango leaves, local mango bark, Kent mango leaves, Kent mango bark, pine needles nor Egyptian date palm seeds supported sporulation of L. theobromae in total continuous light. It took 12 days for pycnidia to form on PDA kept under continuous darkness (Plate 53A) and 25 days to form conidia. The conidia were initially unicellular subovoid to ellipsoidal in shape. Conidia of 15-25 days were bi-celled, thick walled and ellipsoidal in shape (Plate 53C). The rest of the commercially formulated synthetic media (CDB, OGYE, DRBC) did not permit sporulation and therefore no conidia were observed under the microscope (Table 12). The number of conidia per 20-25 microscope fields (high power) recorded on PDA varied from 43 after 15 days to 78 after 25 days; these conidia were averagely 9-12±2.5 µm wide and 20-32±3.2 µm long (Table 12). When the plates were placed under alternating 12 h light and 12 h darkness regime, conidia were formed on PDA and OGYE media only (Plates 54A-C). Conidia formed on DRBC were similar (8.0-12.0±2.0 µm wide and 18.0-30.0±2.5 µm long) to those recorded on PDA (10.0-12.0±1.5 µm wide and 20.0-30.0±2.0 µm long) (Plate 54A & 54B). The immature conidia had initially no septum subovoid to ellipsoidal in shape which later with maturity acquired a single septum (bi- 146 University of Ghana http://ugspace.ug.edu.gh celled) which was thick walled (Plates 54A-54C). The 12 conidia on PDA plates were formed on the 15th day and increased to an average of 42 per plate in 25 days (Table 13). On the other hand, CDA supported the formation of 10 conidia in 20 days in alternating 12 h light and 12 h darkness, and increased only marginally after 25 days (Table 13). Curiously, the natural media, local mango fruit juice agar, Kent (exotic) mango fruit juice agar did not support conidia formation by the mycelium of L. theobromae. However the Soil Extract Agar permitted conidial formation on the medium albeit minimal (Table 14) after 25 days of incubation in alternating 12 h light and 12 h darkness regime. Plate 51 shows the conidia formed in the Soil Extract Agar which is akin to the conidia of the fungus formed in the aforementioned media. The dimension of the conidia 11.0-12.0±3.5 µm wide and 20.0-32.0±4.2 µm long compares favourable with what obtained in PDA and OGYE. 147 University of Ghana http://ugspace.ug.edu.gh P P H A 10µm B 10µm C Plate 53. Sporulation of L. theobromae on Potato Dextrose Agar (PDA) media at 28±1⁰C under continuous darkness for 25 days A=Pycnidium (P) and hyphae (H) on PDA, 22nd day of growth (x100); B= Pycnidia (P) on PDA, 22nd day of growth (x400); C=Matured conidia on PDA, with septum and longitudinal striations (x400) 15µm A 12µm B 16µm C Plate 54. Sporulation of L. theobromae on DRBC and PDA solid media at 28±1⁰C under alternating 12 h light and 12 h darkness for 25 days A=Matured conidia formed in DRBC plate, 25th day of growth; B=Hyphae with immatured non-septate conidia on PDA, 15th day of growth; C=Matured conidia on PDA, 25th day of growth. 148 University of Ghana http://ugspace.ug.edu.gh 10m Plate 55. Sporulation of L. theobromae on Soil Extract Agar at 28±1⁰C under alternating 12 h light and 12 h darkness for 25 days (Note the characteristic ellipsoidal shape of the conidia with the associated hyphae) 149 University of Ghana http://ugspace.ug.edu.gh Table 12. Sporulation of L. theobromae on the indicated commercial synthetic media at 28±1⁰C under continuous darkness for 25 days Average spore Type of Number of spores per 20-25 microscope field views dimension per 20-25 medium (Incubation period in days) microscope fields (µm) 5 10 15 20 25 - - - - - - - - - - - - CDA - - - - - - - - - - - - DBRC - - - - - - - - - - - - - - - - - - - - - - - - PDA - - 43 61 78 9-12±2.5 x 20-32±3.2 - - - - - - OGYE - - - - - - - - - - - - Rep=Replicate; CDA=Czapek-Dox Agar; DRBC=Dichloran Rose-Bengal Chloramphenicol Agar; OGYE=Oxytetracycline Glucose Yeast Extract Agar; PDA=Potato Dextrose Agar; - = Nil. * There are 3 replicates per treatment. 150 University of Ghana http://ugspace.ug.edu.gh Table 13. Sporulation of L. theobromae on indicated selected commercial synthetic media at 28±1⁰C under alternating 12h light and 12 h darkness for 25 days Average spore dimension Type of Number of spores per 20-25 microscope field views per 20-25 microscope fields media (Incubation period in days) (µm) 5 10 15 20 25 - - - - - - - - - - - - CDA - - - - - - - - - 10 11 8-12±2.0 x 18-30±2.5 DBRC - - - - - - - - - - - - - - - - - - - - - - - - PDA - - 12 39 42 10-12±1.5 x 20-30±2.0 - - - - - - OGYE - - - - - - - - - - - - Rep=Replicate; CDA=Czapek-Dox Agar; DRBC=Dichloran Rose-Bengal Chloramphenicol Agar; OGYE=Oxytetracycline Glucose Yeast Extract Agar; PDA=Potato Dextrose Agar; - = Nil. * There are 3 replicates per treatment. 151 University of Ghana http://ugspace.ug.edu.gh Table 14. Sporulation of L. theobromae on mango fruit juice and soil extracts solid media at 28±1⁰C under alternating 12 h light and 12 h darkness for 25 days Type of Number of spores per 20-25 microscope field views Average spore dimension per media (Incubation period in days) 20-25 microscope fields (µm) 5 10 15 20 25 - - - - - - JLF - - - - - - - - - - - - - - - - - - JKF - - - - - - - - - - - - - - - - - - SAE - - - - 2 11-12.0±3.5 x 20-32.0±4.2 - - - - - - JLF=Juice from local mango fruit; JKF=Juice from Kent (exotic) mango fruit; SEA=Soil extract agar ; - = Nil. * There are 3 replicates per treatment. 152 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 20 Pathogenicity test of the causative pathogen of mango tree decline syndrome on host plant Host plants of the mango tree decline disease namely local mango variety and exotic Kent variety were artificially inoculated with the suspected causative pathogen, L. theobromae and were observed for the presence of typical symptoms such as discolouration of vascular tissues, terminal dieback, exudation of gum from stem and bark of intact tissue, browning of affected leaves and upward rolling of the margins of the affected leaves. All the typical symptoms mentioned above were observed on both the local and exotic Kent mango plants after 40 days of incubation in the screen house. Plate 56 shows the typical symptoms of the disease observed on both varieties namely vascular discolouration (Plates 56A & 56B), terminal dieback (Plate 56C), gummosis (Plate 56D) and browning of leaves and upward rolling of their margins (Plate 56E). Plate 53 shows the general view of the symptomatic and asymptomatic plants in the screen house. About 37% of the symptomatic plants showed the tip dieback symptom. Some of the inoculated plants (about three of the local and exotic Kent variety) were asymptomatic suggesting some degree of tolerance of the pathogen especially in the exotic species. Disease incidence (%) and severity index (on a scale of 0-5) was higher (Disease incidence, 71.0%; Severity, 2.0) in the local mango variety than in the Kent exotic variety (Disease incidence, 57.0%; Severity, 1.50) although the severity index of the two varieties were not statistically significant (p˃0.05) (Table 15). To demonstrate the veracity of the pathogen using Koch’s postulate, the fungus L. theobromae was re-isolated from the infected inoculated plants and yielded a recovery of 93.0%. 153 University of Ghana http://ugspace.ug.edu.gh A B I I C D A A Plate 56. A close-up photographs showing I t he typical symptoms of the mango tree decline I disease observed on L. theobromae-inoculated plants in the screened house after 40 days A= Infected twig showing discoloured vascular tissues (arrowed); B= Sliced twig showing discoloured vascular tissue; C=Infected seedling exhibiting terminal dieback (arrowed); D=Exudation of gum (gummosis) from unwounded stem (arrowed) 154 University of Ghana http://ugspace.ug.edu.gh E A Plate 56 (Continued). Symptoms of the mango I d ecline disease observed on seedlings in the screen house E= Browning of affected leaves and upward rolling of their margins 155 University of Ghana http://ugspace.ug.edu.gh A B C D E A A A A A A Plate 57. Photograph showing symptoms of the m ango tree decline dis ease observed on m ango plants in the screen house for 40 days A and B=Control; C and D=Inoculated plants exhibiting browning of leaves and tip dieback (arrowed); E= inoculated plant showing no typical symptom 156 University of Ghana http://ugspace.ug.edu.gh Table 15. Percentage incidence and severity of the pathogenicity test of mango tree decline disease on mango plants in a screen house Mango variety Incidence(%) severity Local 71.00a 2.00a Exotic (Kent) 57.00b 1.50a Mean 64.00 1.75 Means on the same column followed by the same superscript alphabet are not statistically different (p≥0.05) 157 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 21 a. Molecular characterization of the causative agent (L. theobromae) of the mango tree decline disease in Ghana Polymerase chain reaction using species specific primers Approximately, 347 bp product was amplified when the DNA from the isolated pathogen, Lasiodiplodia theobromae, from mango in Ghana were used in a PCR with species specific primer (Lt347-F and Lt347-R). This extracted DNA was successfully identified as Lasiodiplodia theobromae (Plate 58). Polymerase chain reaction using universal primers The universal primers amplified the targeted ITS region of the DNA and the products were successfully sequenced and assembled. The ITS region was approximately 347 bp in size. b. Sequences and phylogenetic analysis of the ITS region An approximately 600 bp product of the ITS region was amplified using the ITS1/ITS4 primer pair. The assembled sequences were 542 bp (Appendix 18). The optimal tree with the sum of branch length = 0.06453193 is shown as Fig. 11. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. All isolates obtained from the mango tree bark clustered together with the type strain of L. theobromae and other L. theobromae isolates with confirmed identities (Fig. 11). All other clades were formed far away from the L. theobromae clade, an indication that the isolates from Ghana were not of those other species. 158 University of Ghana http://ugspace.ug.edu.gh M 1 2 3 4 5 347 bp Plate 58. A gel showing the approximately 347 bp product amplified from Lasiodiplodia species isolated from mango in Ghana Lanes: M=a 1kb marker; 1and 2=DNA from Lasiodiplodia theobromae isolates in Ghana; 3-5=not loaded with any test samples 159 University of Ghana http://ugspace.ug.edu.gh * Fig. 11. Neighbour joining tree drawn with the nucleotide sequences of the ITS region of different species of Lasiodiplodia. The strain identification and species name are provided. Isolates with name preceded by MAN were obtained in this study. Sequences of all other isolates were downloaded from the Genbank. *= Type strain. 160 University of Ghana http://ugspace.ug.edu.gh Table 16. ITS1 grouping of L. theobromae isolates used in the study collected from different locations in Ghana Isolate designation Location of collection MAN-LT1 Dodowa MAN-LT2 Dodowa MAN-LT3 Ayikuma MAN-LT4 Kpong MAN-LT5 Kpong MAN-LT6 Mampong MAN-LT7 Mampong MAN-LT8 Portor 161 University of Ghana http://ugspace.ug.edu.gh EXPERIMENT 22 Preliminary studies on the biocontrol of L. theobromae using the biotoxins of the aqueous extracts of three plants a. Influence of varying concentrations of the aqueous extracts of leaves of Chromolaena odorata, Azadirachta indica and the seeds of Carica papaya on the radial growth of L. theobromae at 28±1⁰C for 72 h The radial growth of the test fungus in the presence of aqueous extracts of all the plants Chromolaena odorata, Azadirachta indica and Carica papaya was commensurate with the concentration of the extract (Figs. 12a-14b). In the PDA amended with aqueous extracts of C. odorata, radial growth in the presence of 1:1 v/v dilution of the extract depressed the growth of L. theobromae by about 50% in 72 h. The pathogen in the extract-free medium covered the plate in 42 h. As dilution increased the biotoxins became less effective (Figs. 12a & 12b). The trend was nearly the same for the pathogen growing on PDA amended with varying dilutions (1:1-1:10 v/v) of the aqueous extract of A. indica. The highest concentration of the biotoxins depressed the vegetative growth of L. theobromae by nearly 50% in 72 h, and there was generally improvement of the growth of the fungus as dilution increased but the growth in dilution up to 1:10 v/v never approximated that of the control plates (Figs. 13a & 13b). The seed extract of C. papaya was less potent and the radial growth which was initially marginally depressed approximated the growth on the control plates after 72 h (Figs. 14a & 14b). 162 University of Ghana http://ugspace.ug.edu.gh Appendices 19-21 show that the difference in radial growth rate (mm/h) are statistically different at p≤0.05. The mycelium did not produce conidia because of the short incubation period of 72 h. However mycelium colour development was same as observed earlier on Plates 5-16. Plates 59-61 show the radial growth of the fungus in different dilution of the test plants. In terms of efficacy in depressing the vegetative growth, one will range the extracts as Chromolaena odorata= Azadirachta indica >Carica papaya. 163 University of Ghana http://ugspace.ug.edu.gh Plate 59. Influence of varying concentrations of the aqueous extracts of Chromolaena odorata leaves on the radial growth of L. theobromae at 28±1⁰C for 3 days From left: Control; 1:10; 1:5; 1:2; 1:1 v/v dilution ratio Plate 60. Influence of varying concentrations of the aqueous extracts of Azadirachta indica leaves on the radial growth of L. theobromae at 28±1⁰C for 3 days From left: Control; 1:10; 1:5; 1:2; 1:1 v/v dilution ratio 164 University of Ghana http://ugspace.ug.edu.gh Plate 61. Influence of varying concentrations of the aqueous extracts of Carica papaya seeds on the radial growth of L. theobromae at 28±1⁰C for 3 days From left: Control; 1:10; 1:5; 1:2; 1:1 v/v dilution ratio 165 University of Ghana http://ugspace.ug.edu.gh 60 50 40 Control 30 1:1 v/v 1:2 20 1:5 10 1:10 0 12 18 24 Period of incubation (h) Fig. 12a. Influence of PDA amended with the indicated concentrations of aqueous extract of Chromolaena odorata leaves on the radial growth of L. theobromae at 28±1⁰C 100 90 80 70 60 Control 50 1:1 v/v 40 1:2 30 1:5 20 1:10 10 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 12b. Influence of PDA amended with the indicated concentrations of aqueous extract of Chromolaena odorata leaves on the radial growth of L. theobromae at 28±1⁰C. 166 Length of mycelia colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh 60 50 40 Control 30 1:1 v/v 1:2 20 1:5 10 1:10 0 12 18 24 Period of incubation (h) Fig. 13a. Influence of PDA amended with the indicated concentrations of aqueous extract of Azadirachta indica leaves on the radial growth of L. theobromae at 28±1⁰C 100 90 80 70 60 Control 50 1:1 v/v 40 1:2 30 1:5 20 1:10 10 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 13b. Influence of PDA amended with the indicated concentrations of aqueous extract of Azadirachta indica leaves on the radial growth of L. theobromae at 28±1⁰C 167 Mean diameter of colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh 60 50 40 Control 30 1:1 v/v 1:2 20 1:5 10 1:10 0 12 18 24 Period of incubation (h) Fig. 14a. Influence of PDA amended with the indicated concentrations of aqueous extract of Carica papaya seeds on the radial growth of L. theobromae at 28±1⁰C 100 90 80 70 60 Control 50 1:1 v/v 40 1:2 30 1:5 20 1:10 10 0 12 18 24 30 36 42 48 54 60 66 72 Period of incubation (h) Fig. 14b. Influence of PDA amended with the indicated concentrations of aqueous extract of Carica papaya seeds on the radial growth of L. theobromae at 28±1⁰C 168 Mean diameter of colony (mm) Mean diameter of colony (mm) University of Ghana http://ugspace.ug.edu.gh b. Vegetative growth of L. theobromae in liquid culture of Potato Dextrose Broth amended with varying concentrations of the aqueous leaf extracts of Chromolaena odorata, Azadirachta indica leaves and the seeds of Carica papaya at 28±1⁰C for 5 Days Vegetative growth by dry matter accumulation by L. theobromae was depressed at higher concentration by the aqueous extracts of the test plants. The higher the concentration of the extract, severer the depression of vegetative growth (Figs. 15-17). C. odorata at 1:1 v/v concentration depressed vegetative growth by nearly 65% (Fig. 15) and further dilution of the extract decreased its potency. Aqueous extract of A. indica at 1:1 v/v dilution decrease dry weight of L. theobromae by about 50% (Fig. 16) and further dilution up to 1:10 v/v decreased the efficacy of the biotoxins. On the other hand, the aqueous extract of C. papaya reduced vegetative growth of L. theobromae by about 40-45% in 5 days but in all instances, the final dry weight obtained never approximated that of the control (Fig. 17). Statistical analysis of the data showed that the difference observed was statistically significant (p≤0.05) (Appendices 22-24). There was no sporulation of the culture since incubation period was too short (5 days) to induce conidia formation. The efficacy of the aqueous extracts can be ranked as follows: Chromolaena odorataCarica papaya (Plates 39-61; Fig. 12-14; Appendices 20-25). Therefore, there is a potential of using plants of medicinal origin to control the fungus. What remains is the identification of the active ingredients and the extension of the incubation time to 21 days when the effect of sporulation can be estimated. Time limitation prevented the suggested work from being carried out. The use of plant extracts for control of pathogenic fungi in Ghana has been well documented (Mensah, 2001; Osei, 1992; Odamtten, 1992; Odamtten and Okyere, 1994; Frimpong, 2007; Wiafe-Kwagyan, 2007; Yasmin et al., 2008; Wiafe- Kwagyan, 2010; Wiafe-Kwagyan and Odamtten, 2013; Agyemang-Boateng, 2016). There is ample evidence that it might be possible to encounter a plant whose active ingredients can be used to effectively control the mango decline fungus. This study extends the list of test plants whose biotoxins can suppress fungal growth in culture in Ghana. This thesis has data to show that the mango decline disease is prevalent in many districts (Berekum, Kintampo, S/Nanton, T/Kumbungu, Kumasi metro, Yilo Krobo, Dangme West, Ga West, Nkoranza, West Gonja, etc.) and Regions (Ashanti, Brong-Ahafo, Eastern, Greater-Accra regions and Northern Regions) in Ghana and is assuming epidemic proportions. The taxonomic characterization of the fungus using cultural, morphological and physiological characteristics show that the disease is caused by L. theobromae in Ghana. The fungus could infect the host mango seedling and produced the symptoms as recorded in the field. This pathogen (L. theobromae) has 184 University of Ghana http://ugspace.ug.edu.gh also been found to infect mango fruits forming a dark-brown lesion which could cover the whole fruit while still hanging on the tree. The disease was identified as a stem end rot by Honger et al. (2014). There is the need to continue this search for biological control using other plants in order to reduce the chemical burden on the environment which has an effect on human and biodiversity alike. If this is done, this thesis would have been a springboard for the management of a major crop disease in Ghana. 185 University of Ghana http://ugspace.ug.edu.gh CONCLUSION AND RECOMMENDATIONS The typical symptoms of the mango decline disease namely tip dieback, shoot dieback, drying of leaves and rolling of their margins, infected branches of the plant becoming discoloured in the vascular tissues while the uninfected branch remained fresh and whitish was observed. As the disease advances, exudates of gum was seen (gummosis) which become heavy as disease severity aggravated. With time the bark cracked as gummosis intensified in severer conditions and callus tissues are formed beneath the bark of the mango tree. In extreme cases of pathogenesis, death of the entire plant is observed. The structured questionnaire showed that majority of farmers (84%) attributed the browning of the foliage to scorching by the sun’s heat and water stress. They (76.2%) also observed abnormal cracking of the bark and vascular browning on infected trees accompanied by oozing of latex from the undamaged portion of the trunk. Still others (57%) recorded the abnormal drying up of twigs and branches and eventual death of the plant. While 4.8% of the respondents attributed the disease to excessive rainfall; 14.3% blamed the disease on poor planting material supplied to them and 4.8% considered poor management practices as responsible for the mango decline disease. A minority, (28.6%) suspected the disease to be caused by an unknown pathogen. The ignorance of the causal agent could aggravate the incidence as erratic and uninformed treatment and application of wrong pesticides may be prescribed by the farmers and extension workers. The farmers identification of the exact symptoms is commendable. Data obtained in this thesis has shown by conventional cultural, physiological and morphological characteristics of the fungus that the causal agent is Lasiodiplodia theobromae. This same fungus 186 University of Ghana http://ugspace.ug.edu.gh was isolated from symptomatic tissue of the host plant could reproduce all the symptoms in artificially inoculated local mango and Kent mango seedlings confirming it to be the true pathogen causing the mango tree decline disease in Ghana. The percentage incidence and severity index was higher on the local mango than the exotic Kent variety. This agree with the field survey data on the two mango varieties (local variety and Kent exotic variety). Further confirmation of the identity of the causative fungus of the mango tree decline syndrome in Ghana was obtained from the molecular characterization studies. PCR studies using specific primers Lt 347-F and Lt 347-R showed that approximately 347 bp products were amplified when the DNA from the pathogen L. theobromae from mango in Ghana were used. This extracted DNA was successfully identified as L. theobromae and the sequences and pathogenic analysis of the ITS region showed that all isolates obtained from the mango tree bark clustered together with the type strain L. theobromae isolates with confirmed identities (Fig. 11). Therefore this confirms the conventional cultural and morphological identification of the causative agent as L. theobromae. This is the first molecular approach in Ghana to successfully identify the local isolates of Lasiodiplodia theobromae. 187 University of Ghana http://ugspace.ug.edu.gh RECOMMENDATIONS 1. This thesis has provided scientific data on the true identity of the causative agent of mango decline disease in Ghana and the farmers have to be educated about this as a first step to curtail spread of the disease. 2. Cultural method of severing disease parts and plugging with clay, etc. may only partially solve the problem. Even when the severed portions are burnt, the fungus can be found in the vascular tissue and in the leaves. 3. There are recommended fungicides for control of mango decline disease caused by L. theobromae. These include Carbendazin (Muhammed et al., 2005), Mancozeb (Sahi et al., 2012), Benomyl, Captan, thiophanate-methyl (Shelar et al., 1997). Farmers in Ghana targeting fresh fruit market place prune the trees with heavy fungicide application (Honger et al., 2014). Some farmers also spray diseased seedlings and young plants showing vascular browning with fungicides every 2 weeks till symptoms disappear. There is the need for further research into the right fungicides to be applied for the control of the pathogen. However, one should bear in mind that sustained spraying with fungicides will increase the chemical burden in the soil and have a detrimental environmental impact eventually. 4a. Biological control using natural plant products presents us with a viable means of controlling plant disease. As a first step, aqueous plant extract of Plectranthus colerides has been shown to depress the vegetative growth of L. theobromae at higher concentration (undiluted, 1:1 v/v dilution) and it prevented sporulation and pycnidial formation (Agyemang-Boateng, 2016). 4b. This thesis provides novel information on the use of aqueous extract of the leaves of C. odorata, Azadirachta indica leaves and Carica papaya seeds to depress in vitro radial and vegetative 188 University of Ghana http://ugspace.ug.edu.gh growth of L. theobromae. The efficacy of the aqueous extracts was ranked in the order Chromolaena odorata=Azadirachta indica Carica papaya. 193 University of Ghana http://ugspace.ug.edu.gh REFERENCES Agrios, G. M. (2005). Plant Pathology. (5th Ed.). Academic Press, New York. 952 pp. Agyeman-Boateng, S. (2016). Preliminary evaluation of the influence of biotoxins of the aqueous extract of Plectranthus coleiodes on vegetative growth of three plant pathogenic fungi of cocoa (Theobromae cacao L.) and mango (Mangifera indica L.). BSc (Hons) Dissertation. Department of Plant and Environmental Biology, University of Ghana, Legon. Pp 38-41. Ahmed, I., Mahmood, A., Majeed, K. and Saleem, A. (1995). Evaluation of various fungicides against dieback disease caused by Diplodia natalensis in mango. Pak. J. Phytopathology, 7: 208- 209. Al-Adawi, A. O., Deadman, M. L., Al Rawahi, A. K., Al Maqbali, Y. M., Al Jahwari, A. A., Al- Saadi B. A., Al-Amri, I. S. and Wingfield, M. J. (2006). Aetiology and causal agents of mango sudden decline disease in the Sultanate of Oman. Eur. J. Plant Pathol., 116: 247-254. Allen, J. (2006). Mango Mania in India. The New York Times.http://www.nytimes.com/2006/05/10/travel/10mumbailetter.html. Retrieved 4 Sept 2013. Alvarez, A. M. and Nishijima, W. T. (1987). Postharvest diseases of papaya. Plant Dis. 71:681-686. Alves, A., Crous, P. W., Correia, A. and Phillips, A. J. L. (2008). Morphological and molecular data reveal cryptic species in Lasiodiplodia theobromae. Fungal Diversity, 28: 1 – 13. Anonymous (2006). Assessment of mango diseases, pests and production problems in Pakistan. Report of a small research activity (SRAhort/2005) on mango in Pakistan. Queensland. 29 pp. Anwar, S. A., McKenry, M. V., and Amad, H. A. (2012). Nematode and fungal communities associated with mango decline of southern Punjab. Pakistan J. Zool., 44:4, pp. 915-922. Arauz, L. F. (2000). Mango Anthracnose: Economic impact and current options for integrated management. Plant Dis., 84 (6):600-611. Arif, A. M., Hussain, N., Ahmad, I., Malik, M. T. and Bally, I. S. E. (2015). Management of mango decline using thiophanate-methyl and plant activators through a macro infusion system. Acta Horticultura, 1105:35-38. http://dx.doi.org/10.17660/ActaHortic. Barreto, J. C., Trevisan, M. T. and Hull, W. E. (2008). Characterization and quantitation of polyphenolic compounds in bark, kernel, leaves, and peel of mango (Mangifera indica L.). J. Agric Food Chem, 56 (14): 5599–610. doi:10.1021/jf800738r. PMID 18558692. Bautista-Baños, S., Barrera-Necha, L. L., Bravo-Luna, I., and Bermudes-Torres, L. (2002). Antifungal activity of leaf and stem extracts from various plant species on the incidence of Colletotrichum gloesporoides of papaya and mango fruit after storage. Rev Mex Fitopatol., 20:8–12. 194 University of Ghana http://ugspace.ug.edu.gh Bicas, J. L., Molina, G., Dionisio, A. P., Barros, F. F. C., Wagner, R., Marostica, M. R. and Pastore, G. M. (2011). Volatile constituents of exotic fruits from Brasil. Food Res Int., 44 (7):1843-1855. Burgess, T. I., Barber, P. A., Mohali, S., Pegg, G., Beer, W. D. and Wingfield, M. J. (2006). Three new Lasiodiplodia spp. from the tropics, recognized based on DNA sequence comparisons and morphology. Mycologia, 98: 423 – 435. Cardoso, J. E., Freire, F. C. O. and Sá, F. T. (1998). Disseminação e controle da resinose em troncos de cajueiro decepados para substituição de copa. Fitopatologia Brasileira, 23: 48–50. Cardoso, J. E., Santos, A. A., Rossetti, A. G. and Vidal, J. C. (2004). Relationship between incidence and severity of cashew gummosis in semiarid north-eastern Brazil. Plant Pathol, 53: 363-367. Carlile, M. J., Watkinson, S. C., and Good, G. W. (2005). The Fungi (2nd Ed.) Elsiever Academic Press, New York. Cilliers, A. J., Swart, W. J. and Wingfield, M. J. (1993). A review of Lasiodiplodia theobromae with particular reference to its occurrence on coniferous seeds. S Afr Forestry, 166(1):47-52. DOI: 10.1080/00382167.1003.9629398. Correl, J. C., Rhoads, D. D. and Guerber, J. C. (1993). Examination of mitochondrial DNA restriction fragment length polymorphisms, DNA finger prints and randomly amplified polymorphic DNA of Colletotrichum orbiculare. Phytopathology 83:1199-1204. Damm, U., Crous, P. W. and Fourie, P. H. (2007). Botryosphaeriaceae as potential pathogens of Prunus in South Africa, with descriptions of Diplodia africana and Lasiodiplodia plurivora sp. nov. Mycologia, 99: 664 – 680. De Candolle, A. P. (1884). Origin of Cultivated Plants, International scientific series, Vol. 49. London, England. Ellis, D. (2016). Lasiodiplodia theobromae. Mycology Online. The University of Adelaide. www.mycology.adelaide.ed.au,2016/Fungal_Descriptions/Coelomycetes/Lasiodiplodia/ El-Zaher, E. H. F. A. (2014). Antifungal activity of Carica papaya seed extract against Aspergillus flavus as serious mycotoxins producing organism and causal organism for aspergillosis. Egypt. J. Exp. Biol. (Bot.), 10 (1):51-62. FAO (1995). FAOSTAT Database Collection. http://apps.fao.org/page/collection. Retrieved November 2015. FAO (2008). FAOSTAT Database Collection. http://.apps.fao.org/page/collection. Retrieved November 2015. 195 University of Ghana http://ugspace.ug.edu.gh FAOSTAT (2000). FAOSTAT Database Collection. Food and cultural organization, production yearbook. Food and Agricultural Organization of the United Nations. Rome, Italy. http://www.fao.org/corp/ statistics/en Accessed 2007 August. FAOSTAT (2009). FAOSTAT Database Collection. http://.apps.fao.org/page/collection. Retrieved November 2015. Fateh, F. S., Kazmi, M. R., Ahmad, I. and Ashraf, M. (2006). Ceratocystis fimbriata isolated from vascular bundles of declining mango trees in Sindh, Pakistan. Pakistan J. Bot., 38:1257–1259. Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39:783-791. Frimpong, L. A. (2007). In vitro studies on the influence of the aqueous leaf extract of Hyptis suaveolens (L) Poit. on some aspects of the physiology of Paecilomyces carneus and Phythophthora palmivora. BSc (Hons) Dissertation. Department of Botany, University of Ghana, Legon. 74 pp. Galinsky, R and Law, N. (1988). World market for mango. RAP Market information Bulletin. No. 9 Gallo, D., Nakano, O., Silveira Neto, S., Carvalho, R. P. L., Baptista, G. C. De; Berti Filho, E., Parra, J. R. P., Zucchi, R. A., Alves, S. B., Vendramin, J. D., Lopes, J. R. S. and Omoto, C. (2002). Entomologia Agrícola. (2nd Ed) Piracicaba: FEALQ. 920 p. Ghana News Agency, GNA (2004). Mango, the next cocoa of Ghana. A news article published by the Ghana News Agency (GNA), Accra, 3 September, 2004. Retrieved December, 2015. http://www.ghanaweb.com/GhanaHomePage/NewsArchive/Mango-to-bring-more-income-to- Ghana-than-cocoa-65446 Hawksworth, D. L., Kirk, P. M., Sutton, B. C. and Pegler, D. N. (1995). Ainsworth & Bisby’s Dictionary of the Fungi. Eighth edition. CAB Int., Wallingford, Oxan OX10 8DE, UK. Hoffman, B. R., Delasalas, H., Blanco, K., Weiderhold, N., Lewis, R.E and Williams, L. (2004). Screening of antibacterial and antifungal activities of ten medicinal plants from Ghana. Pharm Biol, 42(1): 13-17. Honger, J. O., Oduro, K., Offei, S. K. and Odamtten, G. T. (2014). Phenotypic and molecular characterization of the causal agent of mango anthracnose disease in Ghana. Ghana J. Sci, 54:71- 82. Hunter, J. E., and Buddenhagen, I. W. (1972). Incidence, epidemiology and control of fruit diseases of papaya in Hawaii. Trop. Agric. (Trinidad) 49:61-71. 196 University of Ghana http://ugspace.ug.edu.gh Iqbal, Z., Valeem, E. E, Shahbaz, M., Ahmad, K., Khan, Z. I., Malik, M. T. and Danish, M. (2007). Determination of different decline disorders in mango orchards of the Punjab, Pakistan.Pak. J. Bot., 39: 1313-1318. Jason, A. J., Harrington, T. C. and Engelbrecht, C. J. B. (2005). Physiology and taxonomy of the North American clade of the Ceratocystis fimbriata complex. Mycologia, 97(5): 1067-1092. Jedele, S., Hau, M. A., and Von Oppen, M. (2003). An analysis of the world market for mangoes and its importance for developing countries. Paper presented at the Conference on International Agricultural Research for Development. Gottingen, Germany. October 8-10. Jeffries, A., Dodd, J. C., Jeger, M. J. and Plumbley R. A. (1990). The Biology and Control of Colletotrichum species on Tropical Fruit Crops. Plant Pathology, 39:343-366 Jiron, L. F. and Headström, I. (1985). Pollination ecology of mango (Anacardeaceae) in the neotropic region. Turrialba, 35:269-277. Jiskani, M. M. (2002). Mango diseases and their management. Sindhu Agriculture University, Tandojam. www. Pakistan.com/Mango diseases and their management.htm. Johnson, G. I. (1998). Stem end rot p 39-40 in R.C Ploetz, G. A Zetmeyer, W. T. Nishijima, K. G. Rohrbach and H. D. Ohr (eds). Compendium of tropical fruit diseases. The Am Phytopathol Soc, Minnesota. Kazmi, M. R., Fateh, F. S., Majeed, K. and Jabeen, A. (2005).Incidence and etiology of mango sudden death phenomenon in Pakistan. Pakistan J. of Phytopathol., 17(2):154–158. Khalid, P., Akhtar, S. and Alam, S. (2002). Assessment keys for some important diseases of mango. Pak. J. Biol. Sci., 5: 246-250. Khanzada, M. A., Lodhi, A. M. and Shahzad, S. (2004). Mango dieback and gummosis in Sindhu, Pakistan caused by Lasiodiplodia theobromae. Plant Health Progress. DOI:10.1094/PHP-2004- 0302-01-DG. Khuhro, R. D, Nizamani, S. M., Abbasi, Q. D., Solangi, G. S. and Jiskani, M. M. (2005). Mango tree mortality due to Asian ambrosia beetle, Xylosandrus crassiusculus Mot. (Coleoptera: Scolytidae) Pak. J. Agri. Agril. Engg., Vet. Sc., 21(1): 39-42. Kirk, P. M., Cannon, P. F., David, J. C. and Stalpers, J. A. (2001). Dictionary of the fungi. 9th Edition. CABI Publishing. Ko, W. H., Wang, I. T. and Ann, P. J. (2004). Lasiodiplodia theobromae as a causal agent of kumquat dieback in Taiwan. Plant Dis., 88:13-83. Kranz, J. and Rotem, J. (1987). Experimental Techniques in Plant Disease Epidemiology. Springer-Verlag, Berlin. 229 pp 197 University of Ghana http://ugspace.ug.edu.gh Leghari, T. N. (2005). Epidemiology and yield losses by sudden death syndrome in mango orchards of Sindh and its possible control. M. Sc. Thesis. Department of Plant Pathology, SAU Tandojam, pp.124. Leung, B. and Liu, S. (2016). Microbiology 101 Laboratory Manual. Washington State University. http://www.slic2.wsu.edu:82/hurbert/micro101/pages/101lab4.html. Lim, T. K. (1998). Gray Leaf Spot. p 36 in R. C. Ploetz, G. A Zetmeyer, W. T. Nishijima, K. G. Rohrbach and H. D. Ohr (Eds). Compendium of tropical fruit diseases. The Am Phytopathol Soc, Minnesota. Litz, R. E. (1998). Mango; taxonomy description, genetics and breeding and propagation of cultivars, p33-34 in R. C. Ploetz, G. A. Zetmeyer, W. T. Nishijima, K. G. Rohrbach and H. D. Ohr (eds). Compendium of tropical fruit diseases. The Am Phytopathol Soc, Minnesota. Maclean, D. J., Braithwaite, K. S., Manners, J. M. and Irwin, J. A. G. (1993). How do we identify and classify fungal plant pathogens in the era of DNA analysis? Pp. 207-244 In Advances in Plant Pathology 10. Andrews, J. H. and Tommerup, I. C. (Editions). Academic Press, London. Mahmood, A., Saleem, A. and Akhtar, K. M. (2002). Mango decline in Pakistan and its management. Pak. J. Phytopathol., 14: 40-43. Malik, M. T., Dasti, A. A. and Khan, S. M. (2005). Mango decline disorders prevailing in Pakistan. Proceedings of International Conference on Mango and Date palm: Culture and Export, University of Agriculture, Faisalabad, Pakistan. 20-23-June, p. 25. Masood, A., Saeed, S., Dasilveira, S. F., Akem, C. N., Hussain, N. and Farooq, M. (2011). Quick decline of mango in Pakistan: survey and pathogenicity of fungi isolated from mango tree and bark beetle. Pak. J. Bot., 43: 1793-1798. Masood, A., Saeed, S., Malik, M. T., Iqbal, N. and Kazmi, M. R. (2010). Methodology for the evaluation of symptoms severity of mango sudden death disease in Pakistan. Pak. J. Bot., 42: 1289- 1299. Maxwell, L. S. and Betty, M. M. (1984). Florida Fruit. Lewis S. Maxwell, Publisher. pp. 61-63. Mcsorley, R. and Parrado, J. L. (1982). Spatial arrangement of nematodes around four species of tropical fruit trees. Nematropica, 12:247-255. Mcsorley, R., Campbell, C. W., and Goldweber, S. (1980). Observations on a mango decline in south. Proc. Fla. State Hort. Soc. 93:132-133. Mcsorley, R., Campbell, C. W. and Parrado, J. L. (1982). Nematodes associated with tropical and subtropical fruit trees in south Florida. Proc. Fla. State Hort. Soc., 95:132-135. 198 University of Ghana http://ugspace.ug.edu.gh Mensah, J. K. (2001). The use of biofungicides in the control of black pod disease caused Phytophthora palmivora (Butl). Butl. BSc. (Hons) Dissertation. Department of Botany, University of Ghana, Legon. Milne, D. L., Devilliers, E. A. and Holtzhausen, L. C. (1971). Litchi tree decline caused by nematodes. Phytophylactica, 3:37-44. Milne, D. L., Devilliers, E.A. and Van Den Berg, E. (1975). Mango nematodes. Citrus Subtrop. Fruit J., 502:17, 19, 21. Mintz, C. (2008). Sweet news: Ataulfos are in season. Toronto Star Online.Retrieved 1 August 2015. https://www.thestar.com/life/2008/05/24/sweet_news_ataulfos_are_in_season.html Muhammad, A., Aman, U. M., Ahmad, S. K. and Nazir, J. (2005). Potential of fungicides and plant activator for postharvest disease management in mangoes. Int J of Agr. & Biol. ISSN Print: 1560– 8530; ISSN Online: 1814–9596 11–096/AWB/2011/13–5–671–676 http://www.fspublishers.org. Mukherjee, S. K., and Litz R. E. (2009). Introduction: Botany and Importance. The mango: Botany, production and uses. pp. 1-18. Mukherjee, S. K. (1951). Origin of Mango. Indian J. Genet. Pl. Breed.,11: 49–56 Narasimhudu, Y. and Reddy, P. S. N. (1992). A note on gummosis of mango. Indian Phytopathol., 45:261-262. Nimoh, S. E. (2007). A seminar sponsored by the Business Sector Advocacy Challenge (BUSAC) Fund through the Network for Advocacy for Development Alternatives (NADA). www.ghanaweb.com/GhanaHomePage/crime/artikel.php?ID=127908. Article No. 127908. Odamtten, G. T. (1992). The possible role of Chromolaena odorata leaf in the control of Fusarium moniliforme infecting some economic crops. In Chromolaena odorata. Its Biology, Control and Uses. E. Frimpong, Ed. International Council of Scientific Unions / UNESCO / International Biosciences Network Publishers, (ISBN 9964-9-283-8). Pp. 69-76. Odamtten, G.T. and Okyere, G. (1994). The potential use of Tapinanthus bangwensis (Loranthaceae) in the control of Sclerotium rolfsii in Ghana In Aspects of African Mycology. Ed G.L. Hennenebert. International Mycological Association/ Louvanaine-la Neuve, MVCL, Belgium p 125-138. Odebiyi, O. O. (1986). Antimicrobial and antifungal properties of extracts of Jatropha podagrica. Fitoterapia S., 297-299. Oduro, K. A. (2000). Checklist of plant pests in Ghana. Volume 1. Diseases. Plant Protection and Regulatory Service Directorate , Ministry of Agriculture. Accra, Ghana. 105pp. 199 University of Ghana http://ugspace.ug.edu.gh Offei, S. K., Cornelius, E. W. and Sakyi-Dawson, O. (2008). Crop diseases in Ghana and their management. Smartline Publishing Limited. 104 pp. Okigbo, R. N. and Odurukwe, C. N. (2009). Occurrence and control of fungal rot pathogens of yams (Dioscorea spp) with leaf extracts of Chromolena odorata, Carica papaya and Aspilia africana. Niger J myco 2(1):154-165 Osei, P. Y. (1992). Preliminary screening of some local plants for their possible fungitoxic effects on the germination of sporangia of Phytophthora palmivora (Butl). Butl. BSc (Hons) Dissertation. Department of Botany, University of Ghana, Legon. Osuna-Torres, L., Tapia-Pérez, M. E., and Aguilar-Contreras, A. (2005). Plantas medicinales de la medicina tradicional mexicana para tratar afecciones gastrointestinales: Estudio etnobotánico fitoquımico y farmacológico. Universidat de Barcelona, Barcelona. Panhwar, A., Abbasi, Q. D., Nizamani, S. M., Khuhro, R. D. and Rustamani, M. A. (2007). Factors enhancing incidence of mango decline disease complex in Sindhu. 23(2):18-24. http://www.parc.gov.pk/NARC/narc.html. Pavlic, D., Slippers, B., Coutinho, T. A., Gryzenhout, M., and Wingfield, M. J. (2004). Lasiodiplodia gonubiensis sp. nov., a new Botryosphaeria anamorph from native Syzygium cordatum in South Africa. Stud Mycol, 50:313 – 322. Pavlic, D., Wingfield, M. J., Barber, P., Slippers, B., Hardy, G. E. S. J. and Burgess, T. I. (2008). Seven new species of the Botryosphaeriaceae from baobab and other native trees in Western Australia. Mycologia, 100:851 – 866. Ploetz, R. C. (2003). Diseases of Tropical Fruit Crops. In Diseases of mango. (Eds.): R.C. Ploetz. CABI Publisher. p. 327-363. Ploetz, R. C., Benscher, D., Aim, V., Colls, A., Nagel, J. and Schaffer, B. (1997). Mango decline: Research in florida on an apparently widespread disease complex. Abstracts of Presentations on Plant Protection Issues at the Fifth International Mango Symposium, September 1–6, 1996, Tel Aviv, Israel, Phytoparasitica, 25 (1): 45-57. Ploetz, R. C., Benscher, D., Vazquez, A., Colls, A., Nagel J. and Schaffer, B. (1996). A re-evaluation of mango decline in Florida. Plant Dis., 80: 664-668. Popenoe, W., (1920). Manual Of Tropical And Subtropical Fruits. p. 474. The Macmillan Co., New York. Prusky, D. (1998). Alternaria Rot (Black Spot). P. 34-35 in R.C Ploetz, G. A Zetmeyer, W.T. Nishijima, K.G. Rohrbach and H. D. Ohr (eds). Compendium of tropical fruit diseases. The Am Phytopathol Soc, Minnesota. 200 University of Ghana http://ugspace.ug.edu.gh Punithalingam, E. (1976). Botryodiplodia theobromae. CMI Descriptions of Pathogenic Fungi and Bacteria, No. 519. Commonwealth Mycological Institute, Kew, Surrey, England. Ramos, L. J., Lara, S. P., Mcmillan, R.T. and Narayanan, K. R. (1991). Tip dieback of mango (Mangifera indica) caused by Botryodiplodia ribis. Plant Dis.,s 75:315-318. Rawal, R. D. and Ullasa, B. A. (1989). Control of powdery mildew (Oidium mangiferae Berth.) of mango by fungicides. Acta Horticulturae 231(5) DOI: 10.17660/ActaHortic. ISHS Ribeiro, I. J. A. (1980). Seca de manguera. Agentes causais e studio da molesta. In: Anais do I Simposio Brasiliero Sobre a Cultura de Mangeura. Sociedad Brasileira de Fruticultura, Jacoticobal. pp. 123-130. Rocha Ribeiro, S. M., Queiroz, J. H., Lopes Ribeiro De Queiroz, M. E., Campos, F. M. and Pinheiro Sant'ana, H. M. (2007). Antioxidant in mango (Mangifera indica L.) pulp. Plant Foods Hum Nutr., 62(1):13–7. doi:10.1007/s11130-006-0035-3. PMID 17243011. Saeed, S., Khan, M. I. and Masood, A. (2011). Symptom development after artificial inoculation of Botryodiplodia theobromae, a possible causal organism to quick decline in mango trees. Pak. J. agric. Sci.. 48:1-5. Saeed, S. and Masood, A. (2008). Association of Bark beetle Hypocrphalus mangiferae Stebbing (Coleptera: Scolytidae) with Pathogens Ceratocystis fimbriata and Phompsis sp in relation to Mango Sudden Death in Pakistan. International conference, 93rd ESA Annual Meeting, Milwaukee, Wisconsin August 3-8, 2008, USA. Saeed, S., Hussain, N. and Attique, R. (2006). Etiology and management of sudden death phenomenon in mango. Second Annual Report, Department of Entomology, University College of Agriculture, Bahauddin Zakariya University, Multan, pp. 1524. Sahi, S. T., Habib, A., Ghazantfar, M. U. and Badar, A. (2012). In vitro evaluation of different fungicides and plant extract against Botryodiplodia theobromae, the causal agent of quick decline of mango. Pak J. Phytopath., 24(2):137-142. Saksena, N. and Tripathi, S. H. H (1986). Plant volatiles in relation to fungistasis. Fitoterapia, 4(54): 243-244. Saleem, A. and Akhtar, K. M. (1989). Mango diseases and their control. In: Proc. Intl. Mango Workshop Feb. 27 to Mar. 01,1989. (Eds.) Choudhry, A. G. and M. A. Syed). Agri. Ext., Multan, pp. 143-154. Saleem, S., Khanzada, M. A. and Lodhi, A. M. (2006). Mango decline in Sindh. Abstracts, 9th Natl. Conf. of Pl. Scientists 13-15 Feb. 9NCPS-2-16, p. 28. Samson, J. A. (1986). Tropical Fruits. (2nd Ed.) Longman Scientific and Technical. pp. 216-234. 201 University of Ghana http://ugspace.ug.edu.gh Samson, R. A. and Van Reenen-Hoekstra, E. S. (1998). Introduction to Food-Borne Fungi. Centraalbureau Voor Schimmelcultures. Institute of the Royal Netherlands, Academy of Arts and Sciences. 229 pp. Sangeetha, G., Anandan, A., and Usha, R. S. (2010). Morphological and molecular characteristic of Lasiodiplodia theobromae from various banana cultivars causing crown rot diseases in fruits. Phytopathol and Plant Protec, 45(4):475-486. DOI:10.1080/0325408.2011.587986. Sauer, M. R. (1981). Plant nematodes associated with fruit trees in northern Australia. Australian J. Exp. agric. Anim. Husb., 21:129 -131. Schaffer, B., Larson, K. D., Snyder, G. H. and Sanchez, C. A. (1988). Identification of mineral deficiencies associated with mango decline by DRIS. Hort. Sci. J., 23:617-619. Shah, K. A., Patel, M.B., Patel, R. J. and Parmar, P. K. (2010). Mangifera indica (Mango). Pharmacogn, 4(7): 42–48. Sharma, I., (1993). A note on population dynamics and etiology of die back of mango in Himachal Pradesh. New Agricul., 2: 229-230. Shelar, S. A., Padule, D. N., Sawant, D. M. and Konde, B. K. (1997). In vitro evaluation of fungicides against Botryodiplodia theobromae Pat. The cause of dieback disease of mango (Mangifera indica L.). Ind. J. Pl. Proct., 25,118-120. Sial, A. (2002). Mango: A fruit for the world market. Business of Finance Review. The News. pp. 17–19. 10th April 2002, Lahore, Pakistan. Siddiqui, M. A. (2007). Seasonal fluctuation in nematode population associated with mango, Mangifera indica L. Arch. Pl. Path. Pl. Prot., 40:389-394. Subba Rao, N. S. (1977). Soil Microorganisms and Plant Growth, p-251., Oxford and IBH Publishing Co., New Delhi. Susser, A. and Schneider, G. (2001). The Great Mango Book. Ten Speed Press. p. 52. ISBN 1- 58008-204-1. Sutton, B. C. (1980). The Coelomycetes, fungi imperfecti with pycnidia, acervuli and stromata. Commonwealth Mycological Institute. Kew. Tamura, K., Nei, M. and Kumar, S. (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA). 101:11030- 11035. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. (2011) MEGA5: Molecular Evolutionary Genetics Analysis (MEGA) software version 5.0 using Maximum Likelihood, Ev. 202 University of Ghana http://ugspace.ug.edu.gh Úrbez-Torres, J. R., Leavitt, G. M., Guerrero J. C., Guevara J. and Gubler W. D. (2008). Plant Dis., 92(4):519-529, doi:10.1094/PDIS-92-4-0519. USDA National Nutrient Database For Standard Reference (2016). SR-28, Full Report (All Nutrients): 09176, Mangos, raw. National Agricultural Library. USDA. Retrieved 25 January 2016. Uyi O. O., Ekhator, F., Ikuenobe, C. E., Borokini, T. I., Aigbokhan, E. I., Egbon, I. N., Adebayo, A. R., Igbinosa, I. B., Okeke, C. O., Igbinosa, E. O. and Omokhua, G. A. (2014). Chromolaena odorata invasion in Nigeria: A case for coordinated biological control. Manage Biol Inv J, 5(4): 377–393. Vaughan, J. G. and Geissler, C. (1997). The new Oxford Book of Food Plants. Nord. J. Bot 18(2): 202. DOI: 10.1111/j.1756-1051.1998.tb01871.x Vavilov, N. I. (1926). Studies on the Origin of Cultivated Plants. 248 pp. Leningrad. Verma, O. P. and Singh, R. D. (1970). Epidemiology of mango dieback by Botryodiplodia theobromae Pat. Indian J of Afr Sci, 40:813-8. Wiafe-Kwagyan, M. (2007). Evaluation of antifungal activity of oil from nuts of cashew (Anacardium occindentale L.) against Aspergillus flavus, Aspergillus niger and Fusarium verticilloides. BSc. Dissertation. Department of Botany, University of Ghana, Legon. Wiafe-Kwagyan, M. (2010). Influence of some selected medicinal plant extracts on the germination of sporangia and vegetative growth of the Black pod pathogens of cocoa (Phythophthora palmivora (Butl) Bult. and Phythophthora megakarya Brasier and Griffin) and the germination on cocoa (Theobromae cacao L.) seed and development of their seedlings. MPhil. Dissertation, Dept. of Botany, University of Ghana, Legon. Pp 30-31. Wiafe-Kwagyan, M. and Odamtten, G. T. (2013). Influence of some natural plant products on some aspect of the life cycle of the two Phytophthora species causing the black pod disease of cocoa. J Ghana Sci Assoc, 15(2):129-153. Williams, J. G., Ubelik, K., Livak, J., Rafalski, J. A. and Tingey, S. V. (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic acid Res., 18:6531-6535. Woodward, J. E., Langston, B. Jr., Brock, J. H., Kemerait, R. C. Jr., Brenneman, B. And Beard, G. H. (2005). First demonstration of Koch’s postulates for Lasiodiplodia theobromae Fruit spot on eggplant (Solanumme longena). The Am Phytopathol Soc 89 (6): 687. http://dx.doi.org/10.1094/PD-89-0687A. Yasmin, M., Hossain, K. S. and Bashar, M. A. (2008). Effects of some angiospermic plant extracts on in vitro vegetative growth of Fusarium moniliforme. Bangladesh J. Bot., 37 (1): 85-88. Zambettakis, E. C. (1954). Recherches sur la systematique des “Sphaeropsidales-Phaeodidymae”. Bull. Trimest. Soc. Mycol. Fr. 70:219-349. 203 University of Ghana http://ugspace.ug.edu.gh APPENDICES Appendix 1. Questionnaire Title: Knowledge of mango farmers on the recent mango decline disease (syndrome) in Ghana. A. Demographic information of respondents 1. Name of farm/farmers: …………………………………………………………………….. 2. Location of farm (Community): ...…………………………………………………………. 3. Sex a) Male [ ] b) Female [ ] 4. Age a) 20-30[ ] b) 31-40 [ ] c) 41-50 [ ] d) 51-60 [ ] e) 61-70 [ ] 5. Level of education a) No formal education/drop-out [ ] b) Primary/JSS/JHS [ ] c) MSLC [ ] d) SSS/SHS/Voc [ ] B. Background 6. What variety of mango do you grow? a) Keitt [ ] b) Kent [ ] c) Palmer [ ] d) Haden [ ] e) Tommy Atkins [ ] f) Local [ ] g) Others (specify): ……………… 7. Where did you obtain your planting materials? a) Roadside nursery [ ] b) Certified nursery [ ] c) Self [ ] d) Others (specify): ………………………… 8. How old is your plantation? a) 1-5yrs [ ] b) 6-10yrs c) 11-15yrs [ ] d) 15-20yrs e) Other (specify): ……………………………………… 9. What is the size of your plantation? a) < 1ha [ ]b) 1- 2ha [ ] c) 3- 4ha [ ] d) 4-5ha [ ] e) > 5ha [ ] C. Farmer’s knowledge and perception on prevalence of the mango tree decline disease 10. Have you observed any of the following symptoms on any of your mango tree? a) Latex oozing from uncut portions of the trunk [ ] b) Abnormal bark cracking [ ] c) Vascular discolouration (browning) [ ] d) Abnormal drying up and death of branches [ ] e) Branches drying up and breaking off [ ] f) Stunted growth of trees [ ] g) Gradual death of tress [ ] 11. Do you observe these symptoms together on the same trees? Yes [ ] No [ ] 12. If yes, what is the order in which you observed these diseases in reference to question 10 above? ……………………………………………………………………………. 204 University of Ghana http://ugspace.ug.edu.gh 13. What in your view could be the cause(s) of the disease symptoms observed? a) Excessive rainfall [ ] b) Source of planting material [ ] c) Belief and superstition [ ] d) Poor soil [ ] e) Poor management practices [ ] f) Pathogen [ ] g) Wind [ ] h) others (specify)………………………………………………………… 14. When did you first see the symptom(s)? a) Shortly after seedlings were transplanted b) Recently [ ] c) 3-5months ago [ ] d) 6-12months ago [ ] e)1-3yrs ago [ ] f) More than 3yrs ago [ ] 15. Did you initially mistake the browning of leaves as a mere result of sun’s heat and /or water stress? Yes [ ] No [ ] 16. Apart from your farm, have you seen or heard any other farmer complaining about similar disease? Yes [ ] No [ ] 205 University of Ghana http://ugspace.ug.edu.gh 80 70 60 50 40 30 Exotic Local 20 10 0 Ashanti Brong-Ahafo Eastern Greater Northern Accra Administrative region Appendix 2a. Percentage incidence of mango tree decline disease in the different indicated administrative regions of Ghana in 2015 3 2.5 2 1.5 Exotic 1 Local 0.5 0 Ashanti Brong-Ahafo Eastern Greater Northern Accra Administrative region Appendix 2b. Severity of mango tree decline disease in the different indicated administrative regions of Ghana in 2015 206 Mean disease severity % disease incidence University of Ghana http://ugspace.ug.edu.gh 90 80 70 60 50 40 Exotic 30 Local 20 10 0 Coastal Semi-deciduous Transitional Guinea savanna savannah Agro-ecological zone Appendix 3a. Percentage incidence of mango tree decline disease in the indicated four agro- ecological zones of Ghana in 2015 3 2.5 2 1.5 Exotic 1 Local 0.5 0 Coastal Semi-deciduous Transitional Guinea savanna savannah Agro-ecological zone Appendix 3b. Severity of mango tree decline disease in the indicated agro-ecological zones of Ghana in 2015 207 Mean disease severity % disease incidence University of Ghana http://ugspace.ug.edu.gh Appendix 4. Radial growth of L. theobromae on the indicated synthetic media at 28±1⁰C under continuous light (75 lux intensity) for 72 h Period of growth (h) Mean Type Growth of Rep Diameter of mycelia colony (mm) rate media (mm/h) 12 18 24 30 36 42 48 54 60 66 72 ±(SE) 1 4.1 8.7 11.0 15.5 22.0 24.1 27.1 29.8 32.6 38.6 39.0 2 4.4 6.9 12.8 22.1 28.0 31.5 35.7 38.5 43.2 48.2 56.6 0.70±2.09a CDA 3 4.2 6.2 11.4 15.4 22.0 24.9 27.0 29.5 32.2 36.8 55.1 MR 4.2 7.3 11.7 17.7 24.0 26.8 29.9 32.6 36.0 41.2 50.2 1 8.9 15.0 18.7 24.1 29.1 35.8 37.2 43.2 50.0 51.2 59.0 2 9.0 16.0 19.0 25.2 30.0 37.0 39.0 47.0 53.0 58.8 63.0 0.80±0.78a DRBC 3 8.5 15.1 18.0 24.1 28.1 34.0 36.0 42.6 49.0 52.8 58.0 MR 8.8 15.4 18.6 24.5 29.1 35.6 37.4 44.3 50.7 54.3 60.0 1 17.0 31.8 45.0 52.0 64.0 72.0 85.0 90.0 90.0 90.0 90.0 2 18.6 33.1 47.0 56.0 65.6 76.0 89.0 90.0 90.0 90.0 90.0 1.80±0.80b OGYE 3 14.8 30.2 43.1 51.2 62.1 71.0 80.5 88.0 90.0 90.0 90.0 MR 16.8 31.7 45.0 53.1 63.9 73.0 84.8 89.3 90.0 90.0 90.0 1 20.1 30.1 40.2 49.0 60.0 70.5 80.4 90.0 90.0 90.0 90.0 2 19.9 30.2 40.0 50.1 60.8 71.2 80.8 90.0 90.0 90.0 90.0 1.67±0.27b PDA 3 20.5 31.0 42.0 52.2 61.0 72.0 82.4 90.0 90.0 90.0 90.0 MR 20.2 30.4 40.7 50.4 60.6 71.2 81.2 90.0 90.0 90.0 90.0 Rep=Replicate; MR=Mean of replicates; (±)=Standard error; CDA=Czapek-Dox Agar; DRBC=Dichloran Rose-Bengal Chloramphenicol Agar; OGYE=Oxytetracycline Glucose Yeast Extract Agar; PDA=Potato Dextrose Agar. Means in the same column with the same alphabets are not statistically different (p˃0.05). 208 University of Ghana http://ugspace.ug.edu.gh Appendix 5. Radial growth of L. theobromae on the indicated synthetic media 28±1⁰C under constant darkness for 72 h Period of growth (h) Mean Type Growth of Rep Diameter of mycelia colony (mm) rate media 12 18 24 30 36 42 48 54 60 66 72 (mm/h) 1 1.3 6.0 10.0 18.0 26.0 33.0 39.1 45.1 50.9 56.2 60.2 CDA 2 3.0 8.6 15.0 23.1 33.0 40.5 47.7 58.5 63.2 69.2 73.6 a 1.17±0.76 3 2.2 7.2 12.4 21.4 30.0 36.9 44.0 50.5 55.8 63.8 72.1 MR 2.1 7.3 12.5 20.8 29.7 36.8 43.6 51.4 56.6 63.1 68.6 1 5.3 10.7 16.0 22.0 26.8 32.7 39.0 45.7 52.0 58.0 64.0 DRBC 2 5.3 10.9 15.1 21.6 26.9 32.9 41.0 47.0 53.0 59.8 63.0 0.93±0.46b 3 5.9 11.5 18.0 23.5 27.1 33.0 40.0 46.4 56.0 61.8 64.9 MR 5.5 11.0 16.4 22.4 26.9 32.9 40.0 46.4 53.7 59.9 64.0 1 18.1 31.8 45.0 52.0 64.0 72.0 90.0 90.0 90.0 90.0 90.0 OGYE 2 19.6 33.1 47.0 56.0 65.6 76.0 90.0 90.0 90.0 90.0 90.0 1.77±0.88c 3 16.2 30.2 43.1 51.2 62.1 71.0 77.5 90.0 90.0 90.0 90.0 MR 18.0 31.7 45.0 53.1 63.9 73.0 85.8 90.0 90.0 90.0 90.0 1 19.7 28.0 36.0 44.6 54.1 62.4 78.0 83.4 90.0 90.0 90.0 PDA 2 19.7 28.3 37.4 46.0 55.5 64.1 71.0 85.6 90.0 90.0 90.0 1.29±0.89a 3 19.8 36.2 51.0 66.2 80.0 90.0 90.0 90.0 90.0 90.0 90.0 MR 19.7 30.8 41.5 52.3 63.2 72.2 79.7 86.3 90.0 90.0 90.0 Rep=Replicate; MR=Mean of replicates; (±)=Standard error; CDA=Czapek-Dox Agar; DRBC=Dichloran Rose-Bengal Chloramphenicol Agar; OGYE=Oxytetracycline Glucose Yeast Extract Agar; PDA=Potato Dextrose Agar. Values followed by different alphabet = difference statistically significant at p≤0.05. Means in the same column with the same alphabets are not statistically different (p˃0.05). 209 University of Ghana http://ugspace.ug.edu.gh Appendix 6. Radial growth of L. theobromae on the indicated synthetic media 28±1⁰C under alternating 12 h light/12 h darkness regimes for 72 h Type Period of growth (h) Mean of Growth Rep Diameter of mycelia colony(mm) media rate 12 18 24 30 36 42 48 54 60 66 72 (mm/h) 1 7.1 10.7 14.0 17.5 23.0 27.1 31.1 33.8 38.6 42.6 47.0 2 7.4 8.9 15.8 24.1 29.0 34.5 39.7 42.5 49.2 54.2 62.6 0.76±0.60a CDA 3 7.2 8.2 14.4 17.4 23.0 27.9 31.0 33.5 38.2 42.8 61.1 MR 7.2 9.3 14.7 19.7 25.0 29.8 33.9 36.6 42.0 46.5 56.9 1 10.9 13.0 17.7 22.1 27.1 33.8 38.2 43.2 48.0 52.2 56.0 2 11.0 14.0 18.0 23.2 28.0 35.0 40.0 47.0 51.0 57.8 60.0 0.83±0.75a DRBC 3 10.5 13.1 17.0 22.0 26.1 32.0 37.0 42.4 47.0 51.8 55.0 MR 10.8 13.4 17.6 22.4 27.1 33.6 38.4 44.2 48.7 53.9 57.0 1 17.7 24.5 34.0 46.0 58.0 69.9 78.5 88.4 90.0 90.0 90.0 2 16.6 24.1 31.0 39.0 50.6 54.0 63.0 79.0 89.0 90.0 90.0 1.63±0.38b OGYE 3 19.0 28.0 39.1 49.2 59.0 70.0 84.5 90.0 90.0 89.0 90.0 MR 17.8 25.5 34.7 44.7 55.9 64.6 75.3 85.8 89.7 89.7 90.0 1 18.1 28.1 37.2 47.0 58.0 68.5 78.4 84.2 90.0 90.0 90.0 2 17.9 28.2 37.0 48.1 58.8 70.2 78.8 84.4 90.0 90.0 90.0 1.67±0.37b PDA 3 18.5 29.0 39.0 50.2 59.0 70.0 80.4 87.8 90.0 90.0 90.0 MR 18.2 28.4 37.7 48.4 58.6 69.6 79.2 85.5 90.0 90.0 90.0 Rep=Replicate; MR=Mean of replicates; (±)=Standard error; CDA=Czapek-Dox Agar; DRBC=Dichloran Rose-Bengal Chloramphenicol Agar; OGYE=Oxytetracycline Glucose Yeast Extract Agar; PDA=Potato Dextrose Agar Means in the same column with the same alphabets are not statistically different (p˃0.05) 210 University of Ghana http://ugspace.ug.edu.gh Appendix 7. Radial growth of L. theobromae on aqueous extracts of the indicated plant materials at 28±1⁰C under alternating 12 h light/12 h darkness for 72 h Period of growth (h) Mean Type Growth of Rep Diameter of mycelia colony(mm) rate media 12 18 24 30 36 42 48 54 60 66 72 (mm/h) 1 10.6 15.0 20.0 31.9 37.2 40.5 42.5 46.3 53.2 58.1 63.0 2 10.0 14.6 19.7 30.0 37.0 40.0 41.0 45.8 52.0 57.2 62.2 ALB a 3 10.1 14.8 19.9 30.8 37.1 40.1 42.0 46.0 52.8 57.8 62.5 1.19±0.20 MR 10.2 14.8 19.9 30.9 37.1 40.2 41.8 46.0 52.7 57.7 62.6 1 29.4 44.0 57.0 67.2 80.5 90.0 90.0 90.0 90.0 90.0 90.0 2 26.5 40.0 53.0 64.2 79.0 90.0 90.0 90.0 90.0 90.0 90.0 ALL b 3 24.6 36.8 49.0 65.2 78.2 89.0 90.0 90.0 90.0 90.0 90.0 2.17±0.60 MR 26.8 40.3 53.0 65.5 79.2 89.7 90.0 90.0 90.0 90.0 90.0 1 22.7 34.1 50.5 61.2 70.1 80.0 89.0 90.0 90.0 90.0 90.0 2 22.3 34.2 50.0 61.5 73.6 82.0 90.0 90.0 90.0 90.0 90.0 AKB c 3 22.5 33.8 49.0 65.2 70.0 79.5 87.0 90.0 90.0 90.0 90.0 2.14±0.38 MR 22.5 34.0 49.8 62.6 71.2 80.5 88.7 90.0 90.0 90.0 90.0 1 11.0 17.0 20.0 26.0 30.0 35.9 41.0 46.0 50.5 55.0 61.0 2 12.4 18.0 21.0 26.5 31.0 37.0 42.0 47.5 52.0 56.7 63.0 AKL 0.82±0.58d 3 10.0 15.0 19.5 26.0 29.5 34.0 40.0 44.8 49.0 54.0 60.0 MR 11.1 16.7 20.2 26.2 30.2 35.6 41.0 46.1 50.5 55.2 61.3 1 35.0 53.0 71.0 88.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 2 36.0 54.9 71.5 89.3 90.0 90.0 90.0 90.0 90.0 90.0 90.0 2.44±0.46e APN 3 34.0 51.7 69.9 78.0 89.0 90.0 90.0 90.0 90.0 90.0 90.0 MR 35.0 53.2 70.8 85.1 89.7 90.0 90.0 90.0 90.0 90.0 90.0 211 University of Ghana http://ugspace.ug.edu.gh 1 12.0 16.6 23.0 27.0 30.0 35.9 40.0 48.0 54.0 59.5 64.0 2 11.8 15.6 22.0 26.0 29.5 35.0 39.0 46.0 52.0 57.0 62.2 ADS f0.90±0.45 3 12.2 16.0 23.6 27.8 30.0 35.0 40.0 48.0 55.0 61.0 65.0 MR 12.0 16.1 22.9 26.9 29.8 35.3 39.7 47.3 53.7 59.2 63.7 Rep=Replicate; MR=Mean of replicates; (±)=Standard error; ALB=Aqueous local mango bark; ALL=Aqueous local mango leaves; AKL Aqueous Kent mango leaves; AKB=Aqueous Kent mango bark; APN=Aqueous pine needles; ADS=Aqueous Egyptian date palm seeds. Means in the same column with the same alphabets are not statistically different (p˃0.05). 212 University of Ghana http://ugspace.ug.edu.gh Appendix 8. Radial growth of L. theobromae on ethyl acetate extracts of the indicated plant materials at 28±1⁰C under alternating 12 h light/12 h darkness for 72 h Period of growth (h) Mean Type Growth of Rep Diameter of mycelia colony(mm) rate media 12 18 24 30 36 42 48 54 60 66 72 (mm/h) 1 9.0 15.0 22.0 28.5 33.0 38.0 45.0 54.6 61.0 74.0 81.0 2 9.0 14.0 21.0 27.0 34.0 40.0 46.0 55.6 62.0 75.0 82.0 ELB a 3 8.0 14.0 21.0 26.5 32.0 37.0 44.5 53.8 60.0 73.0 80.5 1.11±0.44 MR 8.7 14.3 21.3 27.3 33.0 38.3 45.2 54.7 61.0 74.0 81.2 1 28.4 42.5 55.5 69.2 81.9 90.0 90.0 90.0 90.0 90.0 90.0 2 28.1 42.0 55.0 69.1 81.7 90.0 90.0 90.0 90.0 90.0 90.0 ELL b 3 28.6 42.8 60.0 70.2 82.6 90.0 90.0 90.0 90.0 90.0 90.0 2.10±0.20 MR 28.4 42.4 56.8 69.5 82.1 90.0 90.0 90.0 90.0 90.0 90.0 1 9.0 15.0 22.0 28.5 33.0 38.0 45.0 54.6 61.0 74.0 81.0 2 9.0 14.0 21.0 27.0 34.0 40.0 46.0 55.6 62.0 75.0 82.0 EKB c 3 8.0 14.0 21.0 26.5 32.0 37.0 44.5 53.8 60.0 73.0 80.5 0.75±0.58 MR 9.6 14.3 19.0 24.2 27.7 32.8 37.3 41.3 46.3 55.1 63.4 1 24.0 36.1 51.5 61.1 74.0 86.0 90.0 90.0 90.0 90.0 90.0 2 24.3 36.8 52.0 63.0 75.0 88.2 90.0 90.0 90.0 90.0 90.0 EKL 3 25.5 37.8 52.2 63.2 76.0 88.5 90.0 90.0 90.0 90.0 90.0 1.26±0.20 d MR 24.6 36.9 51.9 62.4 75.0 87.6 90.0 90.0 90.0 90.0 90.0 1 16.0 25.0 43.5 62.1 74.6 86.2 90.0 90.0 90.0 90.0 90.0 2 18.0 26.9 45.5 64.3 76.0 88.0 90.0 90.0 90.0 90.0 90.0 EPN e 3 17.0 25.7 45.0 63.0 75.0 87.0 90.0 90.0 90.0 90.0 90.0 2.48±0.26 MR 17.0 25.9 44.7 63.1 75.2 87.1 90.0 90.0 90.0 90.0 90.0 213 University of Ghana http://ugspace.ug.edu.gh 1 9.0 13.0 21.0 34.5 41.0 61.0 70.0 78.0 86.5 90.0 90.0 2 10.0 15.0 23.0 36.0 43.0 63.0 72.0 79.0 88.0 90.0 90.0 EDS 1.75±0.50f 3 11.0 15.0 23.0 36.5 44.0 64.0 73.0 80.0 89.0 90.0 90.0 MR 10.0 14.3 22.3 35.7 42.7 62.7 71.7 79.0 87.8 90.0 90.0 Rep=Replicate; MR=Mean of replicates; (±)=Standard error; ELB=Ethyl acetate local mango bark; ELL=Ethyl acetate local mango leaves; EKL=Ethyl acetate Kent mango leaves; EPN=Ethyl acetate pine needles; EDS=Ethyl acetate Egyptian date palm seeds. Means in the same column with the same alphabets are not statistically different (p˃0.05). 214 University of Ghana http://ugspace.ug.edu.gh Appendix 9. Radial growth of L. theobromae on petroleum ether extracts of the indicated plant materials at 28±1⁰C under alternating 12 h light/darkness for 72 h Period of growth (h) Mean Type Growth of Rep Diameter of colony(mm) rate media 12 18 24 30 36 42 48 54 60 66 72 (mm/h) 1 6.8 10.0 16.6 20.0 25.0 36.0 41.0 53.6 59.0 64.0 71.0 2 7.0 11.0 17.0 21.0 27.0 38.0 43.0 54.6 62.0 66.0 74.0 PLB a 3 7.0 12.0 18.0 21.5 29.0 39.0 41.5 55.0 63.0 66.0 74.0 1.20±0.58 MR 6.9 11.0 17.2 20.8 27.0 37.7 41.8 54.4 61.3 65.3 73.0 1 8.4 14.5 20.5 29.0 35.9 43.0 50.0 57.0 65.0 72.0 80.0 2 9.0 16.0 21.0 28.0 34.0 42.0 49.0 56.0 65.0 73.0 81.0 PLL b 3 8.0 14.0 21.0 30.0 35.0 42.0 49.0 56.0 64.0 71.0 79.0 1.19±0.38 MR 8.5 14.8 20.8 29.0 35.0 42.3 49.3 56.3 64.7 72.0 80.0 1 8.0 13.0 17.0 21.5 25.0 40.0 45.0 51.6 61.0 66.0 73.0 2 9.0 14.0 18.0 23.0 26.0 40.0 46.0 51.6 62.0 67.0 74.0 PKB c 3 8.0 14.0 18.0 22.5 25.0 40.0 44.5 51.8 61.0 66.0 73.5 1.33±0.25 MR 8.3 13.7 17.7 22.3 25.3 40.0 45.2 51.7 61.3 66.3 73.5 1 9.5 14.0 18.6 26.0 34.0 39.6 58.0 64.0 70.0 77.0 84.0 2 10.0 15.0 19.0 27.0 35.0 41.0 59.0 66.0 71.0 78.0 86.0 PKL c 3 9.0 14.0 18.0 26.0 33.0 38.5 57.0 63.0 70.0 76.0 84.0 1.33±0.44 MR 9.5 14.3 18.5 26.3 34.0 39.7 58.0 64.3 70.3 77.0 84.7 1 15.0 23.0 41.5 60.1 72.6 81.2 90.0 90.0 90.0 90.0 90.0 2 14.0 21.9 43.5 60.3 69.0 76.0 84.0 90.0 90.0 90.0 90.0 PPN d 3 14.0 21.0 41.0 57.0 67.0 79.0 85.0 90.0 90.0 90.0 90.0 1.62±0.61 MR 14.3 22.0 42.0 59.1 69.5 78.7 86.3 90.0 90.0 90.0 90.0 215 University of Ghana http://ugspace.ug.edu.gh 1 11.0 16.0 21.0 32.5 38.0 48.0 55.0 71.0 88.0 90.0 90.0 2 10.0 16.0 22.0 34.0 40.0 49.0 58.0 73.0 89.0 90.0 90.0 PDS 1.74±1.41e 3 11.0 15.0 23.0 36.5 44.0 64.0 73.0 80.0 89.0 90.0 90.0 MR 10.7 15.7 22.0 34.3 40.7 53.7 62.0 74.7 88.7 90.0 90.0 Rep=Replicate; MR=Mean of replicates; (±)=Standard error; PLB=Petroleum ether local mango bark; PLL=Petroleum ether local mango leaves; PKL=Petroleum ether Kent mango leaves; PPN=Petroleum ether pine needles; PDS=Petroleum ether Egyptian date palm seeds. Means in the same column with the same alphabets are not statistically different (p˃0.05). 216 University of Ghana http://ugspace.ug.edu.gh Appendix10. Radial growth of L. theobromae on mango fruit juice and soil extracts at 28±1⁰C under alternating 12 h light/12 h dark regimes for 72 h Period of growth (h) Mean Type Growth of Rep Diameter of colony(mm) rate Media 12 18 24 30 36 42 48 54 60 66 72 (mm/h) 1 20.3 30.4 40.6 50.7 60.8 71.0 87.0 90.0 90.0 90.0 90.0 2 23.6 35.3 47.1 60.0 71.0 82.0 90.0 90.0 90.0 90.0 90.0 JLF 3 24.2 36.2 48.4 61.4 72.0 83.9 90.0 90.0 90.0 90.0 90.0 1.87±0.67 a MR 22.7 34.0 45.4 57.4 67.9 79.0 89.0 90.0 90.0 90.0 90.0 1 18.0 28.0 38.0 47.0 57.0 68.0 77.0 86.5 90.0 90.0 90.0 2 16.0 26.0 35.0 44.0 54.0 65.0 75.5 83.5 89.0 90.0 90.0 JKF 3 17.0 26.0 37.0 46.0 56.0 66.5 76.0 85.0 90.0 90.0 90.0 1.50±0.50b 36. 45. MR 17.0 26.7 55. 7 66.5 76.2 85.0 89.7 90.0 90.0 7 7 1 6.0 9.0 12.0 15.0 18.8 22.7 28.0 31.7 34.0 38.0 42.0 2 6.3 10.9 13.1 16.6 19.9 23.9 30.0 33.0 36.0 40.8 43.0 SEA c 3 5.9 11.5 13.0 16.5 18.1 22.0 28.0 31.4 34.0 38.8 43.9 0.62±0.47 MR 6.6 10.9 13.5 17.0 20.4 24.2 30.7 34.5 37.5 42.4 46.2 Rep=Replicate; MR=Mean of replicates; SE=Standard error; JLF=Juice local mango fruit; JKF=Juice Kent (exotic) mango fruit; SEA=Soil extract agar. Means in the same column with the same alphabets are not statistically different (p˃0.05). 217 University of Ghana http://ugspace.ug.edu.gh 500 450 400 350 300 CDB 250 OGYE-B 200 PDB 150 100 50 0 Period of incubation (4 days) Appendix 11. Dry weight of L. theobromae mycelia in the indicated liquid media at 28±1⁰C under continuous light (75 lux intensity) for 4 days CDB=Czapek-Dox Broth; OGYE-B=Oxytetracycline Glucose Yeast Extract Broth; PDB=Potato Dextrose Broth 218 Mean dry wt. of mycelia (mg) University of Ghana http://ugspace.ug.edu.gh 500 450 400 350 300 CDB 250 OGYE-B 200 PDB 150 100 50 0 Period of incubation (4 days) Appendix 12. Dry weight of L. theobromae mycelia in the indicated liquid media at 28±1⁰C under continuous darkness for 4 days CDB=Czapek-Dox Broth; OGYE-B=Oxytetracycline Glucose Yeast Extract Broth; PDB=Potato Dextrose Broth 219 Mean dry wt. of mycelia (mg) University of Ghana http://ugspace.ug.edu.gh 500 450 400 350 300 CDB 250 OGYE-B 200 PDB 150 100 50 0 Period of incubation (4 days) Appendix 13. Dry weight of L. theobromae mycelia in the indicated liquid media at 28±1⁰C under alternating 12 h light and 12 h dark regimes for 4 days CDB=Czapek-Dox Broth; OGYE-B=Oxytetracycline Glucose Yeast Extract Broth; PDB=Potato Dextrose Broth 220 Mean dry wt. of mycelia (mg) University of Ghana http://ugspace.ug.edu.gh 700 600 500 ALB 400 ALL AKB 300 AKL 200 APN ADS 100 0 Period of incubation (4 days) Appendix 14. Dry weight of L. theobromae mycelia in aqueous extracts of the indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness regimes for 4 days ALB=Aqueous local mango bark; ALL=Aqueous local mango leaves; AKB=Aqueous Kent mango bark; AKL=Aqueous Kent mango leaves; APN=Aqueous pine needles; ADS=Aqueous Egyptian date palm seeds 221 Mean dry wt. of mycelia (mg) University of Ghana http://ugspace.ug.edu.gh 400 350 300 ELB 250 ELL 200 EKB EKL 150 EPN 100 EDS 50 0 Period of incubation (4 days) Appendix 15. Dry weight of L. theobromae mycelia in ethyl acetate extracts of the indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 4 days ELB=Ethyl acetate local mango bark; ELL=Ethyl acetate local mango leaves; EKL=Ethyl acetate Kent mango leaves; EKB=Ethyl acetate Kent mango bark; EPN=Ethyl acetate pine needles; EDS=Ethyl acetate Egyptian date palm seeds 222 Mean dry wt. of mycelia (mg) University of Ghana http://ugspace.ug.edu.gh 400 350 300 PLB 250 PLL 200 PKB PKL 150 PPN 100 PDS 50 0 Period of incubation (4 days) Appendix 16. Dry weight of L. theobromae mycelia in petroleum ether extracts of the indicated plant materials at 28±1⁰C under alternating 12 h light and 12 h darkness for 4 days PLB=Petroleum ether local mango bark; PLL=Petroleum ether local mango leaves; PKB=Petroleum ether Kent mango bark; PKL=Petroleum ether Kent mango leaves; PPN=Petroleum ether pine needles; PDS=Petroleum ether Egyptian date palm seeds. 223 Mean dry wt. of mycelia (mg) University of Ghana http://ugspace.ug.edu.gh 700 600 500 400 JLF 300 JKF SEA 200 100 0 Period of incubation (4 days) Appendix 17. Dry weight and sporulation of L. theobromae in mango fruit juice and soil extracts at 28±1⁰C under alternating 12 h light and 12 h darkness regimes for 4 days JLF=Juice from local mango fruit; JKF=Juice from exotic (Kent) mango fruit; SEA=Soil extract agar 224 Mean dry wt. of mycelia (mg) University of Ghana http://ugspace.ug.edu.gh Appendix 18. ITS1 nucleotide sequences of the 8 isolates of L. theobromae used in the study. >MAN-LT1 TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTTTTCGAGCTTCGGCTCGACTCTCCCACC CTTTGTGAACGTACCTCTGTTGCTTTGGCGGCTCCGGCCGCCAAAGGACCTTCAAACTCCAGTC AGTAAACGCAGACGTCTGATAAACAAGTTAATAAACTAAAACTTTCAACAACGGATCTCTTGG TTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCG TCATTACAACCCTCAAGCTCTGCTTGGAATTGGGCACCGTCCTCACTGCGGACGCGCCTCAAA GACCTCGGCGGTGGCTGTTCAGCCCTCAAGCGTAGTAGAATACACCTCGCTTTGGAGCGGTTG GCGTCGCCCGCCGGACGAACCTTCTGAACTTTTCTCAAGGTTGACCTCGGATCAGGTAGGGAT ACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA >MAN-LT2 TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTTTTCGAGCTTCGGCTCGACTCTCCCACC CTTTGTGAACGTACCTCTGTTGCTTTGGCGGCTCCGGCCGCCAAAGGACCTTCAAACTCCAGTC AGTAAACGCAGACGTCTGATAAACAAGTTAATAAACTAAAACTTTCAACAACGGATCTCTTGG TTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCG TCATTACAACCCTCAAGCTCTGCTTGGAATTGGGCACCGTCCTCACTGCGGACGCGCCTCAAA GACCTCGGCGGTGGCTGTTCAGCCCTCAAGCGTAGTAGAATACACCTCGCTTTGGAGCGGTTG GCGTCGCCCGCCGGACGAACCTTCTGAACTTTTCTCAAGGTTGACCTCGGATCAGGTAGGGAT ACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA >MAN-LT3 TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTTTTCGAGCTTCGGCTCGACTCTCCCACC CTTTGTGAACGTACCTCTGTTGCTTTGGCGGCTCCGGCCGCCAAAGGACCTTCAAACTCCAGTC AGTAAACGCAGACGTCTGATAAACAAGTTAATAAACTAAAACTTTCAACAACGGATCTCTTGG TTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCG TCATTACAACCCTCAAGCTCTGCTTGGAATTGGGCACCGTCCTCACTGCGGACGCGCCTCAAA GACCTCGGCGGTGGCTGTTCAGCCCTCAAGCGTAGTAGAATACACCTCGCTTTGGAGCGGTTG GCGTCGCCCGCCGGACGAACCTTCTGAACTTTTCTCAAGGTTGACCTCGGATCAGGTAGGGAT ACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA 225 University of Ghana http://ugspace.ug.edu.gh >MAN-LT4 TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTTTTCGAGCTTCGGCTCGACTCTCCCACC CTTTGTGAACGTACCTCTGTTGCTTTGGCGGCTCCGGCCGCCAAAGGACCTTCAAACTCCAGTC AGTAAACGCAGACGTCTGATAAACAAGTTAATAAACTAAAACTTTCAACAACGGATCTCTTGG TTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCG TCATTACAACCCTCAAGCTCTGCTTGGAATTGGGCACCGTCCTCACTGCGGACGCGCCTCAAA GACCTCGGCGGTGGCTGTTCAGCCCTCAAGCGTAGTAGAATACACCTCGCTTTGGAGCGGTTG GCGTCGCCCGCCGGACGAACCTTCTGAACTTTTCTCAAGGTTGACCTCGGATCAGGTAGGGAT ACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA >MAN-LT5 TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTTTTCGAGCTTCGGCTCGACTCTCCCACC CTTTGTGAACGTACCTCTGTTGCTTTGGCGGCTCCGGCCGCCAAAGGACCTTCAAACTCCAGTC AGTAAACGCAGACGTCTGATAAACAAGTTAATAAACTAAAACTTTCAACAACGGATCTCTTGG TTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCG TCATTACAACCCTCAAGCTCTGCTTGGAATTGGGCACCGTCCTCACTGCGGACGCGCCTCAAA GACCTCGGCGGTGGCTGTTCAGCCCTCAAGCGTAGTAGAATACACCTCGCTTTGGAGCGGTTG GCGTCGCCCGCCGGACGAACCTTCTGAACTTTTCTCAAGGTTGACCTCGGATCAGGTAGGGAT ACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA >MAN-LT6 TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTTTTCGAGCTTCGGCTCGACTCTCCCACC CTTTGTGAACGTACCTCTGTTGCTTTGGCGGCTCCGGCCGCCAAAGGACCTTCAAACTCCAGTC AGTAAACGCAGACGTCTGATAAACAAGTTAATAAACTAAAACTTTCAACAACGGATCTCTTGG TTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCG TCATTACAACCCTCAAGCTCTGCTTGGAATTGGGCACCGTCCTCACTGCGGACGCGCCTCAAA GACCTCGGCGGTGGCTGTTCAGCCCTCAAGCGTAGTAGAATACACCTCGCTTTGGAGCGGTTG GCGTCGCCCGCCGGACGAACCTTCTGAACTTTTCTCAAGGTTGACCTCGGATCAGGTAGGGAT ACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA 226 University of Ghana http://ugspace.ug.edu.gh >MAN-LT7 TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTTTTCGAGCTTCGGCTCGACTCTCCCACC CTTTGTGAACGTACCTCTGTTGCTTTGGCGGCTCCGGCCGCCAAAGGACCTTCAAACTCCAGTC AGTAAACGCAGACGTCTGATAAACAAGTTAATAAACTAAAACTTTCAACAACGGATCTCTTGG TTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCG TCATTACAACCCTCAAGCTCTGCTTGGAATTGGGCACCGTCCTCACTGCGGACGCGCCTCAAA GACCTCGGCGGTGGCTGTTCAGCCCTCAAGCGTAGTAGAATACACCTCGCTTTGGAGCGGTTG GCGTCGCCCGCCGGACGAACCTTCTGAACTTTTCTCAAGGTTGACCTCGGATCAGGTAGGGAT ACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA >MAN-LT8 TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTTTTCGAGCTTCGGCTCGACTCTCCCACC CTTTGTGAACGTACCTCTGTTGCTTTGGCGGCTCCGGCCGCCAAAGGACCTTCAAACTCCAGTC AGTAAACGCAGACGTCTGATAAACAAGTTAATAAACTAAAACTTTCAACAACGGATCTCTTGG TTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCGGGGGGCATGCCTGTTCGAGCG TCATTACAACCCTCAAGCTCTGCTTGGAATTGGGCACCGTCCTCACTGCGGACGCGCCTCAAA GACCTCGGCGGTGGCTGTTCAGCCCTCAAGCGTAGTAGAATACACCTCGCTTTGGAGCGGTTG GCGTCGCCCGCCGGACGAACCTTCTGAACTTTTCTCAAGGTTGACCTCGGATCAGGTAGGGAT ACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA 227 University of Ghana http://ugspace.ug.edu.gh Appendix 19. A list of isolates downloaded from EMBL Database and used in the study and their GenBank accession numbers ITS region Strain GenBank Species Host Country identification accession numbers Botryodiplodia obtusa CBS 112555 Vitis vinifera Portugal AY259094 Lasiodiplodia Syzygium CBS 115812 S. Africa DQ458892 gonubiensis cordatum Syzygium L. gonubiensis CBS 116355 S. Africa AY639594 cordatum L. missouriana UCD2199MO Vitis vinifera USA JX010251 L. missouriana UCD2193MO Vitis vinifera USA JX010205 L. pseudotheobromae CBS 447.62 Citrus sp. Suriname EF622081 L. pseudotheobromae CBS116459 Gmelina arborea Costa Rica EF622077 L. subglobosa CMM3872 Jatropha curcas Unknown KF234558 L. subglobosa CMM4046 Jatropha curcas Unknown KF234560 L. theobromae CAA 006 Vitis vinifera USA DQ458891 Fruit along coral L. theobromae CBS124.13 USA DQ458890 reef coast Fruit along coral New L. theobromae CBS 164.96* AY640255 reef coast Guinea L. viticola UCD2604MO Vitis vinifera USA HQ288228 L. viticola UCD2553AR Vitis vinifera USA HQ288227 Eucalyptus L. rubropurpurea CBS118740 Queensland DQ103553 grandis *=Type strain 228 University of Ghana http://ugspace.ug.edu.gh Appendix 20. Radial growth of L. theobromae on PDA amended with the indicated varying concentrations of aqueous extract of Chromolaena odorata leaves at 28±1⁰C Dilutions Period of growth (h) Mean of Diameter of colony(mm) Growth Rep extract rate 12 18 24 30 36 42 48 54 60 66 72 (v/v) (mm/h) 1 20.0 30.0 42.0 50.0 62.0 76.5 88.0 90.0 90.0 90.0 90.0 1.94 2 19.0 31.0 42.0 52.0 63.8 78.2 90.0 90.0 90.0 90.0 90.0 ±0.25a Control 3 20.0 31.0 42.0 52.2 64.0 78.0 89.0 90.0 90.0 90.0 90.0 MR 19.7 30.7 42.0 51.4 63.3 77.6 89.0 90.0 90.0 90.0 90.0 1 7.2 10.0 14.4 19.0 24.4 31.0 36.0 41.0 47.0 52.8 57.0 2 7.0 10.0 14.0 20.2 25.0 32.0 36.0 42.0 58.0 53.8 59.0 0.86 1:1 ±0.68b 3 7.0 9.5 13.0 18.1 23.1 30.0 35.0 40.6 46.0 51.0 56.0 MR 7.1 9.8 13.8 19.1 24.2 31.0 35.7 41.2 50.3 52.5 57.3 1 8.0 12.5 19.0 23.0 28.0 36.0 43.0 48.0 52.8 60.0 64.0 2 8.6 13.1 19.0 24.0 29.6 37.0 44.0 49.0 53.0 61.0 65.0 1.02 1:2 ±0.42b 3 8.0 12.0 19.0 23.0 27.0 35.0 42.5 48.0 51.0 58.0 62.0 MR 8.2 12.5 19.0 23.3 28.2 36.0 43.2 48.3 52.3 59.7 63.7 1 10.0 15.0 21.0 26.0 31.0 40.5 47.0 52.0 59.0 65.3 70.0 2 10.0 15.2 21.0 26.5 30.0 40.2 47.0 51.0 59.0 64.0 70.0 1.06 1:5 ±0.13b 3 10.5 15.0 21.0 26.0 31.0 40.0 47.0 51.0 59.0 64.0 70.0 MR 10.2 15.1 21.0 26.2 30.7 40.2 47.0 51.3 59.0 64.4 70.0 1 14.0 21.0 29.0 36.0 44.0 51.0 58.5 64.0 73.0 80.0 86.0 1.25 2 13.0 20.0 28.0 35.2 43.0 50.0 57.5 63.0 72.0 79.0 85.0 ±0.50b 1:10 3 15.0 22.0 30.0 37.2 45.0 52.0 59.5 65.0 73.0 81.5 87.4 MR 14.0 21.0 29.0 36.1 44.0 51.0 58.5 64.0 72.7 80.2 86.1 Means in the same column with the same alphabets are not statistically different (p˃0.05). 229 University of Ghana http://ugspace.ug.edu.gh Appendix 21. Radial growth of L. theobromae on PDA amended with the indicated varying concentrations of aqueous extract of Azadirachta indica leaves at 28±1⁰C Dilutions Period of growth (h) Mean of Diameter of colony(mm) Growth Rep extract rate 12 18 24 30 36 42 48 54 60 66 72 (v/v) (mm/h) 1 20.0 30.0 42.0 50.0 62.0 76.5 88.0 90.0 90.0 90.0 90.0 1.94 2 19.0 31.0 42.0 52.0 63.8 78.2 90.0 90.0 90.0 90.0 90.0 ±0.25a Control 3 20.0 31.0 42.0 52.2 64.0 78.0 89.0 90.0 90.0 90.0 90.0 MR 19.7 30.7 42.0 51.4 63.3 77.6 89.0 90.0 90.0 90.0 90.0 1 8.8 13.0 17.0 21.0 26.0 31.0 36.0 39.0 44.0 50 54.8 0.77 2 9.8 14.0 18.0 23.2 27.0 32.0 37.0 39.0 45.0 51.0 55.8 ±0.50b 1:1 3 7.8 12.0 16.0 20.2 25.0 30.0 35.5 38.0 43.0 49.0 53.8 MR 8.8 13.0 17.0 21.5 26.0 31.0 36.2 38.7 44.0 50.0 54.8 1 9.8 14.0 19.6 24.0 29.0 34.0 40.0 44.4 49.0 55.0 61.0 0.88 2 10.8 15.0 20.6 24.2 30.0 35.0 41.5 45.0 50.0 56.0 62.0 ±0.47b 1:2 3 8.8 13.0 18.6 23.2 28.0 33.0 39.5 43.4 48.0 54.0 60.0 MR 9.8 14.0 19.6 23.8 29.0 34.0 40.3 44.3 49.0 55.0 61.0 1 12.5 18.0 24.0 31.0 37.0 43.0 50.0 57.0 62.0 68.0 73.0 1.08 2 13.5 19.0 25.0 31.2 38.0 44.0 51.5 58.5 63.0 69.0 73.5 ±0.46b 1:5 3 11.5 17.0 23.0 30.2 36.0 42.0 49.5 56.0 61.0 67.0 73.0 MR 12.5 18.0 24.0 30.8 37.0 43.0 50.3 57.2 62.0 68.0 73.2 1 16.5 26.0 35.0 45.0 54.0 66.0 73.0 82.0 89.0 90.0 90.0 1.58 2 15.5 25.0 34.0 44.2 53.0 65.0 72.5 81.0 88.0 89.0 89.0 ±0.47b 1:10 3 17.5 27.0 36.0 45.2 55.0 67.0 74.5 83.0 90.0 91.0 90.5 MR 16.5 26.0 35.0 44.8 54.0 66.0 73.3 82.0 89.0 90.0 89.8 Means in the same column with the same alphabets are not statistically different (p˃0.05). 230 University of Ghana http://ugspace.ug.edu.gh Appendix 22. Radial growth of L. theobromae on PDA amended with the indicated varying concentrations of aqueous extract of Carica papaya seeds at 28±1⁰C Dilutions Period of growth (h) Mean of Diameter of colony(mm) Growth Rep extract rate 12 18 24 30 36 42 48 54 60 66 72 (v/v) (mm/h) 1 20.0 30.0 42.0 50.0 62.0 76.5 88.0 90.0 90.0 90.0 90.0 1.94 2 19.0 31.0 42.0 52.0 63.8 78.2 90.0 90.0 90.0 90.0 90.0 ±0.25a Control 3 20.0 31.0 42.0 52.2 64.0 78.0 89.0 90.0 90.0 90.0 90.0 MR 19.7 30.7 42.0 51.4 63.3 77.6 89.0 90.0 90.0 90.0 90.0 1 11.0 15.0 23.2 28.0 39.0 46.0 52.0 61.0 69.0 81.0 90.0 1.24 2 10.0 14.0 22.2 27.2 38.0 45.0 51.5 60.0 68.0 80.0 89.0 ±0.46b 1:1 3 12.0 16.0 24.2 28.2 40.0 47.0 53.5 62.0 70.0 82.0 90.0 MR 11.0 15.0 23.2 27.8 39.0 46.0 52.3 61.0 69.0 81.0 89.7 1 14.0 19.0 28.0 34.0 44.0 52.0 62.0 71.8 79.0 90.0 90.0 1.47 2 13.0 17.0 27.0 33.2 43.0 51.0 61.5 70.8 78.0 89.0 90.0 ±0.42b 1:2 3 15.0 19.0 29.0 34.2 45.0 53.0 63.5 72.8 80.0 90.0 90.0 MR 14.0 18.3 28.0 33.8 44.0 52.0 62.3 71.8 79.0 89.7 90.0 1 17.5 22.0 34.0 40.0 51.0 60.0 71.0 80.0 86.0 90.0 90.0 1.64 2 16.0 21.0 33.0 40.2 50.0 59.0 70.5 79.0 84.0 89.0 90.0 ±0.43b 1:5 3 18.0 23.0 35.0 41.2 52.0 61.0 72.5 81.0 86.0 90.0 90.0 MR 17.2 22.0 34.0 40.5 51.0 60.0 71.3 80.0 85.3 89.7 90.0 1 19.0 23.0 38.0 47.0 57.0 68.0 76.0 85.0 90.0 90.0 90.0 1.78 2 18.0 22.0 37.0 46.2 56.0 67.0 75.5 84.0 89.0 90.0 90.0 ±0.37b 1:10 3 20.0 24.0 39.0 47.2 58.0 69.0 77.5 86.0 90.0 90.0 90.0 MR 19.0 23.0 38.0 46.8 57.0 68.0 76.3 85.0 89.7 90.0 90.0 Means in the same column with the same alphabets are not statistically different (p˃0.05). 231 University of Ghana http://ugspace.ug.edu.gh Appendix 23. Effect of aqueous extract of Chromolaena odorata leaves on the vegetative growth of L. theobromae at 28±1⁰C for 5 days Dry weight Mean dry wt. of Sporulation per 20- Dilutions of mycelium mycelium ±S.E 25 microscope of extract Rep (mg) (mg) field views (v/v) 1 290.0* Control 2 295.0 291.7±1.44a - 3 290.0 1 95.0 1:1 2 90.0 92.3±1.26b - 3 92.0 1 150.0 1:2 2 150.0 146.7±2.89b - 3 140.0 1 210.0 1:5 2 215.0 211.7±1.44b - 3 210.0 1 250.0 1:10 2 255.0 251.7±1.44b - 3 250.0 Means in the same column followed by different alphabets are significant different at p≤0.05. * Data corrected to the nearest whole number; - = Nil. 232 University of Ghana http://ugspace.ug.edu.gh Appendix 24. Effect of aqueous extract of Azadirachta indica leaves on the vegetative growth of L. theobromae at 28±1⁰C for 5 days Dry weight Mean dry wt. of Sporulation per Dilutions of mycelium mycelium ±S.E(mg) 20-25 microscope of extract Rep (mg) field views (v/v) 1 290.0* Control 2 295.0 291.7±1.44a - 3 290.0 1 160.0 1:1 2 160.0 158.7±1.15b - 3 156.0 1 220.0 1:2 2 220.0 218.3±1.44b - 3 215.0 1 250.0 1:5 2 240.0 245.0±2.50b - 3 245.0 1 260.0 1:10 2 260.0 261.7±1.44b - 3 265.0 Means in the same column followed by different alphabets are significant different at p≤0.05. * Data corrected to the nearest whole number; - = Nil. 233 University of Ghana http://ugspace.ug.edu.gh Appendix 25. Effect of aqueous extract of Carica papaya seeds on the vegetative growth of L. theobromae at 28±1⁰C for 5 days Dilutions Dry weight Mean dry wt. of Sporulation per 20- of extract Rep of mycelium mycelium ±S.E(mg) 25 microscope (v/v) (mg) field views 1 290.0* control 2 295.0 291.7±1.44a - 3 290.0 1 180.0 1:1 2 180.0 180.7±0.58b - 3 182.0 1 230.0 1:2 2 235.0 228.3±3.82b - 3 220.0 1 255.0 1:5 2 250.0 250.0±2.50b - 3 245.0 1 270.0 1:10 2 265.0 266.7±1.44b - 3 265.0 Means in the same column followed by different alphabets are significant different at p≤0.05. * Data corrected to the nearest whole number ; - = Nil. 234