ABSTRACT
Field surveys were conducted during 2014 wet season in three Local Government Areas each of Kaduna, Kano and Katsina States in North West Nigeria to determine the occurrence and distribution of bacterial leaf blight of cocoyam. A total of 81 cocoyam fields were surveyed in the three States. Sampling was done using a five, 2×2 m2 quadrat in each of the nine fields assessed per Local Government Area. Disease incidence recorded in each field showed that the highest incidence of 93.7 % in Kaduna State was obtained in Kudan and the least 16.6 % in Giwa Local Government Areas. In Kano State, the highest 30.0 % and lowest 10.4 % incidence were recorded in Kiru and Bebeji Local Government Areas respectively. With respect to disease severity, Kudan and Giwa Local Government Areas in Kaduna State recorded the highest 45.3 % and the lowest 6.3 % respectively while Kiru and Bebeji Local Government Areas in Kano State recorded the highest 12.7 % and lowest 2.7 % respectively. Bacterial leaf blight was not found in any of the surveyed cocoyam fields in Katsina State. Bacterial isolates obtained from the diseased samples were used for pathogenicity and hypersensitive reaction (HR) tests on cocoyam and tobacco plants respectively and the results indicated that six isolates induced blight symptoms on cocoyam and elicited HR reactions on tobacco. Biochemical tests such as catalase tests, pectolytic tests, amylolytic tests, fluorescence on KB medium and production of acid from glycerol were carried out on the isolates and found that isolates were amylolytic and pectolytic. Furthermore, all the isolates were Gram negative, motile, oxidase negative, catalase positive and did not fluorescent on KB medium and produced no acids from glycerol. For molecular characterisation, a nearly full length of the 16S rDNA gene of selected isolates was PCR amplified using 16S rRNA primers (27F and 1492R) and referenced amplicons were sequenced at the Eurofins Genomics Company, Eurofins MWG
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Operon LLC, Huntsville, Alaska, USA using Sanger sequencing. BLAST results of sequenced genes showed 98-100% homology to Xanthomonas axonopodis pv. dieffenbachiae. Maximum likelihood phylogenetic trees constructed for the 16S rDNA gene sequences revealed isolates were identical to the X. axonopodis pv. dieffenbachiae reference strains AC1 and XADK. Generally, all the isolates obtained were X. axonopodis pv. dieffenbachiae. This is the first report of X. axonopodis pv. dieffenbachiae causing bacterial leaf blight in cocoyam fields in North West Nigeria and the first report on molecular characterisation of the pathogen in Nigeria.
TABLE OF CONTENTS
TITLE PAGE ……………………………………………………………………………………………….. i
DECLARATION …………………………………………………………………………………………. ii
CERTIFICATION ………………………………………………………………………………………. iii
DEDICATION ……………………………………………………………………………………………. iv
ACKNOWLEDGEMENTS …………………………………………………………………………….v
ABSTRACT ……………………………………………………………………………………………….. vi
TABLE OF CONTENTS ……………………………………………………………………………….. i
LIST OF FIGURES ……………………………………………………………………………………….x
LIST OF TABLES ………………………………………………………………………………………. iv
LIST OF PLATES …………………………………………………………………………………………v
LIST OF APPENDICES ………………………………………………………………………………. vi
CHAPTER ONE
1.0 INTRODUCTION ……………………………………………………………………………….1
1.1 Justification of the Study………………………………………………………………………2
1.2 Objectives of the Study…………………………………………………………………………3
CHAPTER TWO
2.0 LITERATURE REVIEW …………………………………………………………………….4
2.1 Origin and Distribution of Cocoyam ……………………………………………………..4
2.2 World Production Trend of Cocoyam ……………………………………………………4
2.3 Importance of Cocoyam ……………………………………………………………………….5
2.4 Constraints to Cocoyam Production in Nigeria ………………………………………7
2.4.1 Abiotic factors ………………………………………………………………………………………7
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2.4.2 Biotic factors ………………………………………………………………………………………..8
2.5 Bacterial Leaf Blight of Cocoyam ………………………………………………………….9
2.5.1 Symptomatology ………………………………………………………………………………… 10
2.5.2 Disease cycle and epidemiology ……………………………………………………………. 10
2.5.3 Host-pathogen interaction …………………………………………………………………….. 11
2.5.4 Economic importance of bacterial leaf blight of cocoyam ………………………….. 12
2.5.6 Characterisation of bacterial leaf blight pathogen …………………………………….. 13
2.5.7 Management of bacterial leaf blight ……………………………………………………….. 16
CHAPTER THREE
3.0 MATERIALS AND METHODS…………………………………………………………. 17
3.1 Incidence and Distribution of Bacterial Leaf Blight of Cocoyam ……………. 17
3.2 Characterisation of Xanthomonas spp. . ………………………………………………. 19
3.2.1 Isolation of the organism, hypersensitive reaction and pathogenicity tests ……. 19
3.2.2 Biochemical tests ……………………………………………………………………………….. 22
3.2.3 Molecular characterisation ……………………………………………………………………. 24
3.3 Statistical Analysis …………………………………………………………………………….. 27
CHAPTER FOUR
4.0 RESULTS ………………………………………………………………………………………… 28
4.1 Incidence and Distribution of Bacterial Leaf Blight ……………………………… 28
4.2 Characterisation of Xanthomonas spp. ……………………………………………….. 35
4.2.1 Tobacco hypersensitive and pathogenicity tests ……………………………………….. 35
4.2.2 Biochemical tests ……………………………………………………………………………….. 38
4.2.3 Molecular characterisation ……………………………………………………………………. 38
CHAPTER FIVE
5.0 DISCUSSION …………………………………………………………………………………… 44
CHAPTER SIX
6.0 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS ……………… 49
6.1 Summary …………………………………………………………………………………………. 49
6.2 Conclusions ………………………………………………………………………………………. 50
6.3 Recommendations …………………………………………………………………………….. 50
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REFERENCES …………………………………………………………………………………………… 51
APPENDICES …………………………………………………………………………………………….. 57
CHAPTER ONE
1.0 INTRODUCTION
Cocoyam (Colocasia and Xanthosoma spp.) belongs to the family Araceae. It is one of the oldest crops grown mostly in the tropics, for its edible corms, cormels and leaves and as an ornamental plant (Adelekan, 2012). Cocoyam is one of the basic food crops of major economic importance and ranks third in importance after cassava and yam among the root and tuber crops cultivated and consumed in Nigeria (Baruwa and Oke, 2012). It is superior to yam and cassava nutritionally, with higher protein, mineral nutrients and vitamin contents and its starch is more readily digested (Odebunmi et al., 2007). On a global scale, it ranks 14th as a vegetable crop with annual production figure of over 9 million tonnes (FAOSTAT, 2014). Africa accounts for at least 60% of world production and most of the remaining 40% is from Asia and Pacific regions (Mitra et al., 2007). Nigeria is the world‟s leading producer of cocoyam, with annual production of 3.27 million metric tonnes, accounting for about 36 percent of total world output of cocoyam (FAOSTAT, 2014). Cocoyam is commonly grown for its starchy edible roots. Colocasia sp. is grown for its corm and cormels which are consumed after boiling, frying or roasting. The corms can be dried and used to make flour or sliced and fried to make chips. The leaves of the plant are also edible and are usually consumed as a vegetable after cooking in dishes such as stews. Xanthosoma sp. produces tubers much like potato and are boiled, baked, steamed or fried prior to consumption. Young leaves are eaten as a vegetable (Azeez and Madukwe, 2010).
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The yield and quality of this crop are threatened by various abiotic and biotic factors. Diseases caused by fungi and bacteria are among biotic factors militating against the production of cocoyam in Nigeria. The diseases include phytophthora leaf blight caused by Phytophthora colocasiae (Bandyopadhyay et al., 2011; Zarafi et al., 2012), corm rot induced by some species of fungi such as Aspergillus niger, Fusarium solani, Fusarium oxysporum and Sclerotium rolfsii (Ugwuoke et al., 2008), corm soft rot induced by Pectobacterium spp. (Amodu and Akpa, 2012) and the bacterial leaf blight (BLB) of cocoyam induced by Xanthomonas axonopodis pv. dieffenbachiae (Opara et al., 2013).
1.1 Justification of the Study
Xanthomonas axonopodis pv. dieffenbachiae (Xad) is considered the most serious bacterial pathogen causing extensive crop losses in commercial foliage plant production worldwide because of its virulence and broad host range within the Araceae family (Donahoo et al., 2013). Bacteria leaf blight of cocoyam was first reported in Nigeria at Umudike, South-Eastern Nigeria where the crop was found to be highly susceptible to the pathogen (Opara et al., 2013). Reports of cocoyam diseases from farmers‟ fields in Kaduna State prompted a preliminary survey of some cocoyam fields in Kaduna State where bacterial leaf blight was observed among symptoms of diseases threatening cocoyam production in the State. The existence of Xad in North West Nigeria became highly probable due to transfer of planting materials among cocoyam growers and climate change. There was the need to assess the status of this disease in North West Nigeria where the crop occupies a very important position in the farming system. The crop is grown to fill hunger period and to provide instant cash to the farmer, with an average yield of 7000 kg/ha (NAERLS and NAPFS, 2010).
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Efficient management of this pathogen requires a sensitive and reliable diagnostic tool (Robene-Soustrade et al., 2006). A review of available literature has shown that limited reports on the identity of the pathogen in Nigeria have been focused on biochemical method of identification (Opara et al., 2013). This method, apart from being time consuming, is highly limited in species differentiation. Molecular characterisation of Xad using Polymerase chain reaction (PCR) based technique has proven to be a fast, sensitive and reliable method for determining genetic relationships among pathogenic organisms (Khoodoo and Jaufeerally-Fakim, 2004; Robene-Soustrade et al., 2006). This technique has become indispensable for quarantine purposes and effective breeding for resistance to bacterial diseases in order to achieve desirable disease management. This research will provide valuable information that will form the basis for cocoyam bacterial leaf blight disease management decisions and breeding cocoyam cultivars with resistance to the pathogen. Molecular characterisation of the pathogen in the region will also provide information that could be used to map the pathogen in the region, a tool that could serve quarantine purposes.
1.2 Objectives of the Study
The objectives of this study are to:
1) determine the incidence and distribution of bacterial leaf blight of cocoyam in Kaduna, Kano and Katsina States
2) characterise isolates of bacterial leaf blight pathogen.
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