ABSTRACT
Field survey was conducted in 2015 between August and September in three Local Government Areas of Kaduna, Katsina and Kano States of Nigeria to determine the incidence, distribution and alternative hosts of Jatropha Mosaic Virus on Physic nut (Jatropha curcas L). A total of twenty seven (27) Jatropha curcas farms were surveyed in three States (Kaduna, Katsina and Kano), nine in each State. Sampling was done in three demarcated 12 × 12 m2 plot in each of the selected fields per Local Government Area. Jatropha curcas and other plant species showing virus like symptoms which included mosaic, leaf curl, mottling, stunting, chlorosis, yellowing, and necrotic symptoms as well as asymptomatic plants were sampled. A total of twenty eight weed samples were randomly collected from the three States. Analysis was carried out for the detection of viral components (DNA-A and B) and sub-viral components (DNA-β and 1) of Jatropha mosaic virus by Polymerase Chain Reaction (PCR). Jatropha mosaic virus disease was present in all farm locations and the incidences recorded. The highest disease incidence in Katsina State (95 %) was recorded at Walin-Hausa Foundation farm (Tsanni) in Batagarawa LGA, and the lowest (43.75%) in Katarda of Batagarawa LGA. In Kaduna State, the National Research Institute for Chemical Technology (NARICT) Jatropha curcas field had the highest incidence (83.61%), with the lowest (42.86%) at Gwaraji in Igabi LGA. The highest disease incidence (88%) recorded in Kano State was at Marmara in Tudun-Wada LGA, and lowest (2%) in the Institute for Agricultural Research (I.A.R) sub-station Jatropha curcas farm at Minjibir LGA. The viral components DNA-A and B were not detected in all the fields across the three States, as well as the sub-viral component DNA-1 while the sub-viral component DNA-β was detected only on some samples from NARICT and I.A.R Jatropha
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curcas fields both in Sabon-Gari Local Government Area of Kaduna State. The helper virus component (DNA-A) was not detected. None of the plant species samples tested positive to any of the viral and sub-viral components of interest in this study from the three States. This is the first report of a satellite virus DNA-β of JMV in Kaduna State of Nigeria. In view of the importance of Jatropha curcas L. in Nigeria, threats such as Jatropha mosaic virus attack to its production should be considered seriously and prevention as well as management strategies be pursued vigorously.
TABLE OF CONTENTS
TITLE PAGE ……………………………………………………………………………………………………….. i
DECLARATION …………………………………………………………………………………………………. ii
CERTIFICATION ………………………………………………………………………………………………. iii
DEDICATION ……………………………………………………………………………………………………. iv
ACKNOWLEDGEMENTS ……………………………………………………………………………………. V
ABSTRACT ………………………………………………………………………………………………………. vi
TABLE OF CONTENTS ……………………………………………………………………………………. viii
LIST OF TABLES ………………………………………………………………………………………………. xi
LIST OF FIGURES…………………………………………………………………………………………….. xii
LIST OF PLATES ………………………………………………………………………………………………xiv
CHAPTER ONE
1.0 INTRODUCTION …………………………………………………………………………………………….1
1.1 Justification of study………………………………………………………………………………………….5
1.2 Objectives of study ……………………………………………………………………………………………6
CHAPTER TWO
2.0 LITERATURE REVIEW …………………………………………………………………………………..7
2.1 Jatropha Classification ……………………………………………………………………………………….7
2.2 Origin and Distribution of Jatropha………………………………………………………………………7
2.3 Production of Jatropha curcas …………………………………………………………………………….9
2.4 Economic Importance of Jatropha……………………………………………………………………… 11
2.5 Pests and Diseases of Jatropha ………………………………………………………………………….. 15
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2.6 Jatropha Viruses …………………………………………………………………………………………….. 16
2.6.1 Cucumber mosaic virus (CMV) genus Cucumovirus ………………………………………….. 16
2.6.2 Jatropha Mosaic Virus. ………………………………………………………………………………… 16
2.7Criteria for Diagnosis of viruses ………………………………………………………………………… 17
2.7.1Symptomatology of Jatropha mosaic virus ………………………………………………………… 17
2.7.2Host range of Jatropha mosaic virus ………………………………………………………………… 19
2.7.3Detection of Jatropha mosaic virus ………………………………………………………………….. 20
2.7.3.1 Polymerase chain reaction (PCR) …………………………………………………………………. 20
2.8Management of Jatropha mosaic virus ………………………………………………………………… 22
CHAPTER THREE
3.0 MATERIALS AND METHODS ………………………………………………………………………. 24
3.1 Survey and Sample Collection ………………………………………………………………………….. 24
3.2 Determination of Alternative Hosts of the Virus ………………………………………………….. 25
3.3 Detection of Jatropha Mosaic Virus …………………………………………………………………… 25
3.3.1 DNA Extraction…………………………………………………………………………………………… 25
3.3.2 Polymerase Chain Reaction procedure …………………………………………………………….. 30
3.3.3 Agarose gel electrophoresis …………………………………………………………………………… 35
CHAPTER FOUR
4.0 RESULTS …………………………………………………………………………………………………….. 36
4.1 Incidence of Jatropha Mosaic Virus …………………………………………………………………… 36
4.2 Polymerase Chain Reaction ……………………………………………………………………………… 41
4.3 Alternate hosts of Jatropha mosaic virus …………………………………………………………….. 53
CHAPTER FIVE
5.0 DISCUSSION ……………………………………………………………………………………………….. 54
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CHAPTER SIX
6.0SUMMARY, CONCLUSION AND RECOMMENDATIONS ……………………………….. 57
6.1Summary ……………………………………………………………………………………………………….. 58
6.2 Conclusion ……………………………………………………………………………………………………. 59
6.3 Recommendations ………………………………………………………………………………………….. 68
REFERENCES APPENDICES
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background to the Study Jatropha curcas L.often referred to as „Jatropha‟ is one of the prospective oil-yielding plants with vast industrial potential as bio-diesel (Jayanna, 2006). The seeds can be pressed into bio-oil that has good characteristics for direct combustion in compressed ignition engines or for the production of bio-diesel (Putten et al., 2010). The bio-oil can also be a raw material for soap making. The pressed residue of the seeds (pressed cake) is a good fertilizer and can also be used for bio-gas production. Jatropha curcas grows under subtropical conditions and can withstand conditions of severe drought and low soil fertility. It is capable of growing in marginal soil, and it can help to reclaim problematic lands. It is not a forage crop, hence, it plays an important role in deterring cattle as the leaves are not palatable, and thereby, protecting other valuable food or cash crops when used in fencing (Putten et al., 2010). Current interest by farmers and non-governmental organizations (NGOs) in Jatropha curcas is mainly due to its potential as an energy crop.
Jatropha curcas grows in tropical and sub-tropical regions, with cultivation limits at 30ºN and 35ºS (FACT, 2007). It also grows in lower altitudes of 0-500 meters above sea level. It is not sensitive to day length (flowering is independent of latitude) and may flower at any time of the year (Heller, 1996). It is a succulent shrub that sheds its leaves during the dry season, with deep roots that make it well suited to semi-arid conditions. While Jatropha can survive with as little as 250 to 300 mm of annual rainfall, at least 600 mm is needed for flowering and fruit setting (FACT, 2007). The optimum rainfall for seed production is
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considered between 1000 and 1500 mm (FACT, 2007), which corresponds to sub-humid ecology. Higher precipitation is likely to cause fungal attack and restrict root growth in all but the most free-draining soils (cited by Achten, 2008).Jatropha curcas is not found in the more humid parts of its area of origin, Central America and Mexico. Rainfall induces flowering and, in areas of unimodal rainfall, flowering is continuous throughout most of the year (FACT 2007). Optimum temperatures are between 20ËšC and 28ËšC. Very high temperatures can depress yields (Gour, 2006). Jatropha curcas is intolerant of frost. The plant is well adapted to conditions of high light intensity (Baumgaart, 2007) and is unsuited to growing in shade. Insect pests and diseases pose a significant threat to Jatropha curcas. Observations of free-standing older trees would appear to confirm this, but incidence of insect pests and diseases is widely reported under monoculture plantation, and may be of economic significance. The Jatropha curcas plants suffer from several diseases, which include damping off, root rot, leaf spots, frog-eye leaf spot, powdery mildew and Jatropha mosaic virus disease. Observed diseases, such as collar rot, leaf spots, root rot and damping-off, may be controlled with a combination of cultural techniques (for example, avoiding waterlogged conditions) and fungicides (Achten, 2008). Jatropha multifida is a known host of African cassava mosaic virus as well as a possible source of transmission of the cassava super-elongation disease caused by Sphaceloma manihoticola (Achten, 2008).
The family Geminiviridae comprises of a group of plant infecting circular ssDNA viruses that severely constrain agricultural production throughout the World, and a particular serious threat to food security in sub-Saharan Africa. Worldwide, they are responsible for a
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considerable amount of economic damage to many crops such as cassava, sweet potato, tomatoes, cowpea, okra, cotton, grain legumes and now Jatropha curcas (Fauquet et al., 2008). Geminiviruses are distinct in having circular, single-stranded DNA (ssDNA) genomes that are encapsidated within twinned icosahedral virions. Displaying substantial diversity in terms of their primary nucleotide sequences, genome structures, host ranges and insect vectors, the family Geminiviridae has been divided into seven different genera. Besides the Begomoviruses, these include the genera Mastrevirus, Curtovirus, and Topocuvirus (Fauquet et al., 2008) and three additional genera Becurtovirus, Eragrovirus and Turncurtovirus (Varsani et al., 2014).
The genus Begomovirus with over one hundred and ninety two recognized species, contains more species than all the other geminivirus genera combined (Fauquet et al., 2008; Brown et al., 2011). Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component) or bipartite (two circular ssDNA components called DNA-A and DNA-B), many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-1/α) or betasatellites (DNA-β) (Marie et al., 2012). Geminiviruses are often associated with sub-viral agents called DNA satellites that require proteins encoded by the helper virus for their replication, movement and encapsidation. Hitherto, most of the single-stranded DNA satellites reported to be associated with members of the family Geminiviridae have been associated with monopartite begomoviruses. These satellite molecules completely lack sequence identity to their helper viruses and depend on the helper virus for all or some of the following functions: replication, movement, encapsidation and transmission. Satellite molecules were initially
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reported to be associated with RNA viruses; these satellites are very well characterized (Simon et al., 2004). In the last decade, more than 500 satellite sequences associated with begomoviruses (family Geminiviridae) have been isolated from a diverse range of cultivated crops and weeds (Briddon and Stanley, 2006; Briddon et al., 2008). Alphasatellites are 1.3 kb nanovirus-like components that, in some cases, suppress viral disease symptoms. Although alphasatellites encode a replication associated protein, they depend on the helper virus encoded proteins for movement and encapsidation (Briddon and Stanley, 2006; Nawaz-ul-Rehman and Fauquet, 2009). Betasatellites are a diverse set of symptom-enhancing, single-stranded DNA (ssDNA) molecules that are 1.3 kb in size and only associated with monopartite begomoviruses from the Old World (OW), namely Asia and Africa (Briddon et al., 2001, 2003, 2008). Most recently, betasatellites have been found associated with a few bipartite begomoviruses (Rouhibakhsh and Malathi, 2005).
Cook in 1931 described the occurrence of Jatropha mosaic disease for the first time in Puerto Rico (USA). Jatropha mosaic virus was first reported on Jatropha gossypiifolia (Bird, 1957) as a euphorbiaceous weed prevalent throughout the West Indies. Several viruses have been found to affect Jatropha curcas. Kashina et al. (2013) reported a complete nucleotide sequence of a begomovirus (Jatropha Mosaic Nigerian Virus) naturally infecting Jatropha curcas in Nigeria which is a threat to the realization of the full potential of the crop. The symptoms observed were severe mosaic, mottling and blistering of leaves. Pair-wise comparisons of DNA-A sequences showed that Jatropha mosaic virus had maximum nucleotide sequence identity (72 %) with a strain of Tomato yellow leaf curl virus. Ramkat et al. (2011) reported the occurrence of cassava geminiviruses on Jatropha curcas grown in
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Kenya. Raj et al. (2008a) reported the incidence of Cucumber mosaic virus on Jatropha curcas in India. Snehi et al. (2008) identified a new begomovirus on J. gossypifolia in India.
1.2 Justification of the study
The crude oil crisis of the 1970s and the subsequent shortages of petrol-fuels in the world market have brought to light the limitations of world oil resources (Grimm, 1996; Heller, 1996; Henning, 2000; Pratt et al., 2002). Currently, the world is faced with critical fuel shortages accompanied with high prices, as well as, the global warming issue. This has prompted government, scientists and non-governmental organizations (NGOs) to search for alternative sources of energy, which are renewable and environmentally safe. In this regard, renewable vegetable fuels have assumed top priority. Special interest has been shown in the cultivation of the tropical physic nut (Jatropha curcas L., Euphorbiaceae) for oil extraction (Grimm 1996; Heller 1996).Worldwide, Jatropha is gaining more prominence considering its potential as an energy and biofuel crop. Although much of this potential has not been harnessed fully in Nigeria, elsewhere it has been demonstrated that the crop can help solve many problems ranging from land reclamation, bio-diesel production and protection of valuable crops such as cash or food crops from cattle (Jayanna, 2006). The potential yield, quality and value of the croparelowered by pest attack reducing the quality of seeds produced, thereby, affecting its potential of fulfilling the promise of an energy crop. Recently, virus diseases have posed a great threat to the crop‟s potential due to their insidious nature, which usually results in the development of epidemics, if not managed properly.
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Most of the plant viruses have weeds or other alternative natural hosts that provide a reservoir of viruses from which the economically important crops may become infected (Neeraj and Zaidi, 2008; Mathews and Dodds, 2008). Weeds, ornamental plants and wild plants near crop fieldsare infected with the viruses of cultivable plant species (Sivalingam and Varma, 2007) and play a key role in the development of virus disease epidemics, because the bio trophic nature of plant viruses obviously requires the alternate hosts to continue association of virus-host-vectors association for the sustainability of the viral pathogens in nature in the absence of original crop host. All these make the virus disease epidemiology more complex and hazardous as compared to other diseases (Maramorosch and Harris, 1981). Identification of initial source of infection is important for studying the epidemiological aspects of plant viruses and also to formulate control measures for the viral diseases (Thresh, 1982). The expected benefit of the proposed research is that knowledge about the existence, distribution and alternative hosts of the virus in Nigeria will aid the development of effective management strategies to enhance the growth of jatropha and its success as a potential energy crop and a renewable energy source.
1.3 Objectives of the study
This research was undertaken to fulfill the following objectives:
1. To determine the incidence and distribution of Jatropha mosaic virus (JMV) in some Northwestern States of Nigeria.
2. To identify the alternative hosts of the virus.
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