ABSTRACT
Cassava mosaic virus is said to occur in all growing areas in Africa and a substantive land area is devoted to cassava in Kano and Katsina State of Nigeria. There is also the need to investigate if Cassava congo sequivirus recently reported in Kaduna and Sokoto States occurs in the States. Field surveys were conducted during the 2016 dry and wet seasons in three local government areas each of Kano and Katsina States of Nigeria to determine the occurrence, distribution and alternative hosts of viruses of cassava (Manihot esculenta Crantz). A total of 18 cassava farms were surveyed in the twoStates (nine in each State). Sampling was done in 5 x 5 m2 quadrats in each field of the three selected fields per Local Government Area. Cassava plants with mosaic, distortion, stunting, chlorosis and necrotic symptoms on leaves, as well as, those with no obvious virus disease symptoms were sampled. A total of twenty weed samples, were also collected during both dry and wet seasons andanalysis was carried out for the presence of cassava viruses. Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA) was used to detect Cassava congo sequivirus(CCSV) while the Triple Antibody Sandwich Enzyme Linked Immunosorbent Assay (TAS-ELISA) was used to detect African cassava mosaic virus(ACMV) and East african cassava mosaic virus (EACMV). After the analyses, three Cassava viruses Cassava congo sequivirus, African cassava mosaic virusand East african cassava mosaic virus were detected either singly (ACMV 30.4 %, EACMV 21.5 %, CCSV 17.8 %) (ACMV 28.9 %, EACMV 25.2 %, CCSV 28.1 %) or in mixed infections of two or even the three viruses (ACMV + EACMV 11.1 %, ACMV + CCSV 2.2 %, EACMV + CCSV 1.5 % and ACMV + EACMV + CCSV 1.5 %) (ACMV + EACMV 9.6 %, ACMV + CCSV 14.8 %, EACMV + CCSV 5.9 % and ACMV + EACMV + CCSV 3.0 %) in all the Local Governments Areas of
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Kano and KatsinaStates respectively. This provides the first report of these viruses in the States and the second report of the occurrence of CCSV in Nigeria. None of the weeds could be confirmed as hosts of the viruses.
TABLE OF CONTENTS
COVER PAGE TLE PA …………………………………………………………… Error! Bookmark not defined.
DECLARATION ………………………………………………………………………………………………………………. ii
CERTIFICATION …………………………………………………………………………………………………………….. iii
DEDICATION …………………………………………………………………………………………………………………. iv
ACKNOWLEDGEMENTS …………………………………………………………………………………………………. v
ABSTRACT …………………………………………………………………………………………………………………….. vi
TABLE OF CONTENTS………………………………………………………………………………………………….. viii
LIST OF FIGURES ……………………………………………………………………………………………………………. x
LIST OF TABLES ……………………………………………………………………………………………………………. xi
LIST OF PLATES ……………………………………………………………………………………………………………. xii
LIST OF APPENDICES…………………………………………………………………………………………………… xiii
CHAPTER ONE ……………………………………………………………………………………………………………….. 1
1.0 INTRODUCTION ………………………………………………………………………………………………………… 1
1.2 Justification of the Study ………………………………………………………………………………………….. 3
1.3 Objectives ……………………………………………………………………………………………………………….. 4
CHAPTER TWO……………………………………………………………………………………………………………… 5
2.0 LITERATURE REVIEW ………………………………………………………………………………………….. 5
2.1. The Cassava Plant ………………………………………………………………………………………………………… 5
2.1.1 Botany of cassava ……………………………………………………………………………………………………… 5
2.1.2 Origin of cassava ………………………………………………………………………………………………………. 6
2.1.3 Cassava production …………………………………………………………………………………………………… 6
2.1.4 Cultivation of cassava ……………………………………………………………………………………………….. 8
2.1.5 Nutritional composition of cassava ……………………………………………………………………………… 8
2.1.6 Uses of cassava …………………………………………………………………………………………………………. 9
2.1 Constraints to Cassava Production ………………………………………………………………………………. 10
2.3 Cassava Viruses ………………………………………………………………………………………………………… 11
2.3.1 African cassava mosaic virus, East african cassava mosaic virus and South African cassava mosaic virus ……………………………………………………………………………………………………………………. 11
2.3.2 Cassava brown streak disease (CBSD) …………………………………………………………………………. 13
2.3.3 Cassava congo sequivirus ………………………………………………………………………………………….. 14
2.3.4 Alternative hosts of cassava viruses ………………………………………………………………………….. 15
2.3.5 Economic importance of cassava viruses …………………………………………………………………… 15
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2.4 Diagnosis of cassava viruses …………………………………………………………………………………………. 16
2.4.1 Serological tests ………………………………………………………………………………………………………. 16
2.4.2 Molecular tests ……………………………………………………………………………………………………….. 17
CHAPTER THREE ………………………………………………………………………………………………………….. 18
3.0. MATERIALS AND METHODS ………………………………………………………………………………….. 18
3.1 Survey and Sampling of Cassava Fields ……………………………………………………………………….. 18
3.1.1 Disease incidence …………………………………………………………………………………………………….. 19
3.2 Detection of Cassava Mosaic Virus Using Enzyme-linked Immunosorbent Assay (ELISA) Techniques ……………………………………………………………………………………………………………………… 22
3.2.1 Triple Antibody Sandwch Enzyme-Linked Immunosorbent Assay (TAS-ELISA) For the Detection of ACMV and EACMV in Cassava leaves ……………………………………………………………… 22
3.2.2 DAS-ELISA for the detection of cassava congo sequivirus (CCSV) in cassava leaf samples …. 23
CHAPTER FOUR ……………………………………………………………………………………………………………. 25
4.0 RESULTS …………………………………………………………………………………………………………………. 25
4.1 Symptoms of Cassava Viruses in the Fields ………………………………………………………………. 25
4.2 Incidence of the three Cassava Viruses in Katsina State ………………………………………………… 25
4.3 Incidences of the three cassava Viruses in Kano State ………………………………………………… 27
4.4 Distribution of Viruses Infecting Cassava in Katsina and Kano States. …………………………… 29
4.5 Distribution of Viruses Infecting Cassava in Kano and Katsina States. ……………………….. 31
4.6 Alternative hosts of Cassava Viruses in Katsina and Kano States ……………………………….. 36
CHAPTER FIVE ……………………………………………………………………………………………………………… 38
5.0 DISCUSSION ……………………………………………………………………………………………………………. 38
CHAPTER SIX ……………………………………………………………………………………………………………….. 43
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS……………………………………………. 43
6.1 Summary ………………………………………………………………………………………………………………. 43
6.2 Conclusion …………………………………………………………………………………………………………….. 44
6.3 Recommendations ………………………………………………………………………………………………….. 44
REFERENCES………………………………………………………………………………………………………………… 45
APPENDICES ………………………………………………………………………………………………………………… 56
CHAPTER ONE
1.0 INTRODUCTION
Cassava, Manihot esculenta Crantz, is a perennial woody shrub with an edible root, which grows in tropical and sub-tropical areas of the world (IITA, 2009). It is widely cultivated for its tuberous root that is rich in carbohydrate. It belongs to the family Euphorbiceae that also includes other commercially important plants like castor bean (Ricinus communis L.) and rubber (Havea bransiliensis L.). Cassava and some 90 other species make up the genus Manihot and it is the only widely cultivated member of this genus. It is the third most important source of calories in the tropics, after rice and maize. More than 276 million tonnes of cassava was produced worldwide in 2016, of which Africa accounted for 56 % (FAOSTAT, 2017). Nigeria produced 57.1 million tonnes making it the world’s largest producer (FAOSTAT, 2017). It provides over 500 calories daily to over 70 million people (Westby, 2008).
Cassava crop is afflicted with many insect pests and diseases including cassava spider mite, cassava mealy bug(Phenacoccus manihoti), green mite (Mononychellus tanajoa), African cassava mosaic disease, cercospora leaf spot disease, cassava bacterial blight, anthracnose, cassava brown streak diseas, and nematodes (particularly Meloidogyne spp.) among others (and Ortiz, 2007). African cassava mosaic disease (ACMD) is the most severe and widespread disease caused by viruses limiting production of the crop in sub-Saharan Africa. Although Africa is the largest producer of cassava, yields in Africa are very low (estimated at 8.9 tonnes/ha) as compared to other cassava producing regions (Asia and Latin America) despite the fact that, under optimal conditions cassava can produce up to 80 tonnes/ha of roots in a 12- month culture period (Nassar and Ortiz, 2007). A number of factors are responsible for
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the severely reduced yields in Africa, and the most important constraints are virus diseases, particularly cassava mosaic disease (CMD), cassava brown streak disease (CBSD) as well as bacterial blight (caused by Xanthomonas axonopodis pv. manihotis). Other constraints are poor agricultural practices and various other diseases caused by bacteria, fungi and nematodes (Hillocks and Wydra, 2002), most of which are considered of minor importance. The most important single reason for this is probably the almost ubiquitous presence of cassava mosaic disease (CMD) (Dixon et al., 2003; Legg et al., 2006). CMD is caused by a group of cassava mosaic geminiviruses (CMGs) (Geminiviridae: Begomovirus). They comprise twinned (geminate) particles, approximately 20-30nm in size, containing a bipartite, single stranded, circular DNA genome (Harrisonet al., 1977).
Prior to 1995, only two African species of CMDs were recognised, Africancassava mosaic virus(ACMV) and East african cassavamosaic virus, and serology-based diagnostic techniques were used to show that the two had largely non-overlapping distributions, in which ACMV occurred throughout West, Central and much of southern Africa, whilst EACMV was largely confined to coastal East Africa, Malawi, Zimbabwe and Madagascar (Swanson and Harrison, 1994). As nucleic acid-based diagnostics were introduced in the later part of the 1990s, however, much greater complexity was revealed. A novel EACMV/ACMV recombinant was described from Uganda (EACMV-UG) (Zhou et al., 1997; Deng et al., 1997), South african cassava mosaic virus(SACMV) was reported from southern Africa (Berry and Rey, 2001) and three additional EACMV species were described (Fauquet and Stanley, 2003). These includedEast african cassava mosaicCameroon virus (EACMCV), East africa cassava mosaicMalawi virus (EACMMV) and East African cassava mosaic zanzibar virus (EACMZV). Additionally, mixed ACMV+EACMV virus infections were shown to be
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common, frequently giving rise to synergistic interactions and enhanced symptom severity (Harrison et al., 1997). ACMV and EACMV were shown to co-occur in most areas where CMD occurred (Ogbe et al., 1999; Legg and Thresh, 2000; Berry and Rey, 2001). Further diagnostics and characterization work seems certain to reveal the presence of an even greater diversity of CMGs and CMG interactions (Legg and Owor, 2003). In cassava fields, yield losses due to CMD can be as high as 95.0 % (Fauquet and Fargette, 1990). Cassava plants infected with CMGs express a range of symptoms which depend on the virus species/strain, environmental conditions, and the sensitivity of the cassava host. The most typical symptoms consist of a yellow or pale green chlorotic mosaic of leaves, commonly accompanied by distortion and crumpling. Symptoms are readily distinguished from those of mineral deficiency or cassava green mite damage as the virus induced chlorosis and malformation of leaflets is asymmetrical about the midrib. Two main methods are used to manage CMD. They include the use of CMD resistant or tolerant varieties and the implementation of sanitation techniques. The latter method consists of using only cuttings from healthy plants for planting and subsequently removing any infected plants (Ntawuruhunga et al., 2007).
1.2 Justification of the Study
Cassava (Manihot esculenta Crantz) is an important staple food crop of nearly a billion people in the world ( Mbanzibwa et al., 2011). It is the third largest carbohydrate food source within the tropical regions, after rice and corn (Ceballos et al., 2004). It is referred to as a food security crop (Barrett et al., 2006). Africa produces more than half of the world‟s cassava production. Although Nigeria is reported to be the highest producer of cassava, the average yield realized per hectare (6.2t/ha) (NAERLS, FDAE and P& PCD, 2017) is still far less than
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what is obtained elsewhere e. g India (37t/ha). The reason for such more than 50 % yield loss may not be unconnected to the presence of insect pests and diseases (most especially virus diseases). African cassava mosaic virus is said to occur in all the areas where cassava is grown in Africa. Most of the work on cassava viruses in Nigeria was done in Southern guinea, derived Savannah zone and rain forest zones, however,not much work has been reported in Northern guinea and Sudan savannah zones. Over the years, there has been an increase in the production and consumption of cassava in the North West zone of Nigeria. This may not be without an increase in the incidence of cassava diseases. Kano has been reported to have the lowest average yield per hectare in this zone (NAERLS and FDAE, 2014). There is scanty information on weed hosts of cassava viruses particularly in Nigeria. Information on alternative hosts is an important tool in the disease management. Preliminary survey conducted in Kano and Katsina States revealed symptoms of CMD. It is also not clear if CCSV very recently reported in Kaduna and Sokoto States (Badamasi, 2017) is present in Kano and Katsina States. Therefore, it is necessary to carry out this research in Kano and Katsina States.
1.3 Objectives
The objectives of this research are to: 1. Determine the occurrence and distributionof cassava virusesin Kano and Katsina States. 2. Identify the alternative hosts of the viruses.
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