COLLEGE OF PLANT SCIENCE AND CROP PROD.UCTION UNIVERSITY OF AGRICULTURE, ABEOKUT A, OGUN STATE, NIGERIA.

Transcription

1 COLLEGE OF PLANT SCIENCE AND CROP PROD.UCTION UNIVERSITY OF AGRICULTURE, ABEOKUT A, OGUN STATE, NIGERIA.

2 This is to certify that this project was carried out by THOMAS, Hea 0 Department Dr. O. R. Pitan QL~~.d~!~_ Date Supe " or Dr. A. A. 'Fajlnmi B.Se (llorin), M.Se, Ph.d (Ibadan) (r ~ (J.ZOf () --- L_i ---- _. Date

3 This project is dedicated to Almighty God for His infinite mercy, guidance and protection throughout the programme, and to the Department of Crop Protection, UNAAB

4 I ascribe all the glory, honour and adoration to the Almighty God for his guidance and protection throughout my stay on this Campus and in the Department of Crop Protection. My profound gratitude goes to my dynamic project supervisor, Dr. A.A. Fajinmi for his guidance, encouragement and for sparing his time to make all necessary correction in my project work and to make sure I use best of the best facilities for laboratory analysis. My heartfelt regard goes to my loving parents. Mr. and Mrs. F.A. THOMAS for endless care and support, may God Almighty continue to bless you and also spare your lives so that you will both eat the fruit of thy labour. My genuine appreciation goes to my siblings, Emmanuel, Mary, Martha for their love and moral support, God will continually bless you in all ramifications. A lot of thanks to a loving brother, Afam, who stood by me financially during my early days in UNAAB, I pray that God will grant you an unequal favor. I will not fail to say a big thank you to my friends Ore, Dara, Tayo, Micheal, Bunmi, Kehinde, Idris, may God see you all through your endeavors.

5 Cowpea (Vigna unquiculala L. Walp) is an important leguminous crop in the world. Among the viral diseases that affect cowpea, Cowpea Aphid Borne Mosaic Virus [CABMy] is considered to be a major viral diseases and affects large scale production of this crop. Using grow out potted experiment followed by Antigen Coated Plate Enzyme-Linked Immuno Sorbent Assay [ACP-ELISA] based testing of the seedlings mechanically inoculated with CAB MY, sap extract collected from International Institute of Tropical Agriculture, (IITA), ten varieties of cowpea seeds sourced from, Institute of Agricultural Research and Training, (lar&t) and five varieties sourced from a local market, [Bodija market at Ibadan], were grown in pots. Harvested seeds from the CABMy infected potted plants were subjected to a grow out test and the subsequent seedlings that emerged expressed characteristic symptoms of interveinal chlorosis, leaf distortion, stunting and this was also confirmed through ACP- ELISA serological assay. Only the five varieties (IT , BROWN BERRET, IFE- BPC, IFE 98-12, IFE BROWN) out of the ten varieties sourced from Institute for Agricultural Research and Training, (IAR&T) were confirmed negative for the virus. This showed that CABMy is a seed-borne virus disease therefore there is need to screen cowpea seeds before being sold to farmers for planting in order to reduce the spread of CABMy and its subsequent economic effect. Reason for the five varieties sourced from IAR&T not expressing the symptom of C ABMy cannot be confirmed by this experiment.

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8 Contents Certification Dedication Acknow ledgements Abstract Table of Contents List of Tables List of Plates Page II III IV V VI vii VIII CHAPTER ONE 1.0 Introduction CHAPTER TWO 2.0 Literature Review 4 CHAPTER THREE 3.0 Materials and Methods 8 CHAPTER FOUR 4.0 Results 12 CHAPTER FIVE 5.1 Discussion Conclusion Recommendation 16 References 17

9 Cowpea (Vigna unquiculata L. Walp) is one of the world's dicotyledonous leguminous food crops and a major food crop of millions of people in the developing countries (Summerfield et ai., 1974). Cowpea belongs to the kingdom plantae, division (Magnoliophyta), class (MagnolIopsida), order (Fabales), family fabaceae formally (Leguminiseae), Sub-family (Faboideae), genus (Vigna) and species (unquiculata). Cowpea has probably been used as a crop plant since Neolithic time. A lack of archaeological evidence has resulted in contradicting views supporting Africa, Asia and South America as its origin (Summerfield et ai., 1974; Coetzee, 1995). One view is that cowpea was introduced from Africa to the Indian sub- continent approximately 2000 to 3500 years. Cowpea provides an extremely significant portion of the dietary protein of the people and plays an important nutritional role in developing countries of the tropics and subtropics especially in sub-saharan Africa (Rachie, 1985). Cowpea young leaves, pods and pea contain vitamins and minerals which have fuelled its usage for human consumption and animal feeding (Rachie et ai., 1985). In the United States, green seeds are sometimes roasted like peanuts and consumed. The roots of the cowpeas are eaten in Sudan and Ethiopia and the scorched seeds are occasionally used as a coffee substitute (Duke, 1987). In Nigeria, cowpeas are used to make soups and bean mixes such as "moi.-moi" and beans cakes. The leaves of cowpea may be boiled, drained, sun-dried and then stored for later use (Duke, 1987). This world's dicotyledonous crop is highly variable crop 2100 Afr. 1. Biotechnol cultivated around the world essentially for the seeds and also as vegetables in which there are about fifteen varieties of cowpea in common cultivation (Duke, 1987). Nigeria is reputed to be

10 the highest producer of cowpea in the world (Taiwo and Akinjogunla, 2006). Some other countries like Niger, Mail, Brazil and Australia produce significant amount. This leguminous food crop is greatly attacked by wide array of diseases of biological origin especially viruses which cause devastating effects and are a really constraint to increased yield of cowpea in several countries (Ladipo 1977 et at). Viral diseases have become serious due to extensive cultivation of cowpeas and also viral diseases have significant status because they do not only cause direct damage to the host, but they equally predispose the plants to secondary invader. 1.1 Economic Importance of Viral Diseases of Cowpea The effects of viruses can be devastating and are a major constraint to the production of cowpea (Thottappilly and Rossel, 1992). Resultant effect of cowpea viral diseases such as poor pod formation and quality has been a major constraint in cowpea production in most part of the world. Diseases of plant viruses affect some parts of cowpea plants including the leaves, foliage, pods and young seedlings. (Thottappilly and Rossel, 1992). The production of cowpea is constrained by a vast array of pathogenic organism and insect. Infection can result in loss of saleable produce from the plant (Hughes, 2003). Infection by several viruses occurs in cowpea production which reduces its yield and quality of pod. 1.2 Cowpea Virus Diseases Over 140 viruses have been reported worldwide to infect cowpea cultivars but only nine have been reported in Nigeria, and these are Cowpea aphid-borne mosaic virus (CABMV), genus Potyvirus; Cowpea golden mosaic virus (CPGMV), genus Bigeminivirus; Southern bean mosaic virus (SBMV), genus Sobemovirus; Sunhemp mosaic virus (SHMV), genus Tobamovirus;

11 Blackeye mosaic virus (BICMV), genus Potyvirus; Cucumber mosaic virus (CMV), genus Cucumovirus; Cowpea mottle virus (CMeV), genus Carmovirus; Cowpea yellow mosaic virus (CPMV) genus, Comovirus; Cowpea mild mottle virus (CPMMV), genus Carlavirus. (Shoyinka, 1974; Hughes et ai2003). During some seasons, plants may be infected with only one virus, while in the other seasons plants are infected with a combination of viruses. Both quality and quantity of yield can be significantly reduced in severely infected field (Taiwo, 2006). Transmission of plant viruses could either be persistent or non-persistent manner [Akinjogunla, 2005]. Aphid borne viruses that are transmitted non-persistently are most abundant and wide spread of all known plant viruses, they comprise of several taxonomic group represented by the following genera; Potyvirus, calavirus, Alfamovirus, Fabavirus, Caulimovirus and Cucumovirus, (Taiwo, 2003). The majority of the viral diseases of cowpea lead to overall stunting, reduction in leaf size, mottling, mosaic, leaf chlorosis, leaf distortion, leaf curling, vein clearing, necrotic local lesion and death (Akinjogunla, 2005). 1.3 Objective of the study To investigate the transmission of cowpea aphid borne mosaic virus (CABMV) in grown out seedling of cowpea.

12 CHAPTER LITERATURE TWO REVIEW 2.1 Diseases of Cowpea Viral diseases of cowpea (Vigna unguiculata), varies in terms of the severity depending on host cultivar and virus strain. O. Diseased cowpea plants show variable amounts of dark green vein banding or interveinal chlorosis, leaf distortion, blistering and stunting. Viruses that may be related cause mosaic diseases of adzuki bean (Phaseolus angularis) and asparagus bean (Vsesquipedalis). (Adegbite, and Amusa, 2008) Incidence of viral diseases on cultivated cowpea Cowpea is greatly attacked by wide array of diseases of biological origin especially viruses which cause devastating effects and are a real constraint to increased yield of cowpea in several countries (Adegbite, 2008). Of all the known viruses of cowpea, cowpea aphid-borne mosaic virus is responsible for the major viral disease of cowpea in Nigeria, (Shoyinka et ai., 1997). In reference to a three year survey for the incidence and distribution of cowpea viruses, detected viruses in 390 out of 649 cowpea collected from all agro ecological zones in Nigeria using ELISA, CABMV had the highest incidence and was the most prevalent of all the viruses detected, (~hoyinka et ai., 1997) Symptoms of viral diseases of cowpea, The majority of the viral diseases of cowpea lead to overall stunting, reduction in leaf size, i<lliottling,mosaic, leaf chlorosis, leaf distortion, leaf curling, vein clearing, necrotic local lesion

13 and death (Akinjogunla, 2005). Typical symptoms of viral infections are stunting, mottling or foliage and deformed pod. 2.2 Cowpea aphid borne mosaic virus (CABMV) Cowpea aphid borne mosaic virus (CABMV), a member of the Potyvirus genus, a distinctive virus with flexuous filamentous particles c. 750 nm long. It is seed-borne in cowpea, has a wide experimental host range, is transmitted by several common species of aphid, and occurs in many countries where cowpea is grown, (Bock, 1974) It causes a severe mosaic of cowpea (Vigna unguiculata), the severity depending on host cultivar and virus strain. Diseased cowpea plants show variable amounts of dark green vein banding or interveinal chlorosis, leaf distortion, blistering and stunting, (Thottapilly and Rossel, 1992) Viruses that may be related cause mosaic diseases of adzuki bean (Phaseolus angularis) and asparagus bean (V. sesquipedalis), (Tsuchizaki et al 1970). It occurs in Africa (Kenya, Uganda and Nigeria), Europe (Italy, and probably Rumania), and Asia (India, Iran, Japan, and probably China), viruses that are probably related occur in the USA (Florida) and in the south-west Pacific area, (Bock et al1974). It can cause a yield loss of 13-87% under field conditions depending upon crop susceptibility, virus strain and the environmental conditions, (Taiwo, 2003). CABMV has spread world-wide through the exchange of virus-infected germplasm material. The virus-infected seed provides the initial inoculum and aphids are responsible for the secondary spread of the disease under field conditions, (Taiwo, 2003). The virus symptoms vary with the cowpea genotype and virus strain. Excellent sources of resistance are available for the breeding of resistant cultivars. Resistance in cowpea is conferred by either a dominant or a recessive gene, (Thottapilly and Rossel, 1992).

14 2.2.1 Spread of viral Diseases on Cultivated Cowpea Viruses can be transmitted by insects, primarily Aphids, leafhoppers, and thrips (Taiwo, 2006). Sometimes whiteflies can transmit viruses (Taiwo, 2006). Mites, fungi and nematodes can occasionally transmit viruses (Shoyinka, 1997). However, there are two types of viral transmission which could be persistent or non-persistent. By a vector, the virus is transmitted in the stylet-borne, non-persistent manner by Aphis craccivora (Bock, 1973), A. fabae, A. gossypii, A. medicaginis, Macrosiphum euphorbiae and Myzus persicae ( Taiwo, 2006). Non-feeding aphids can retain infectivity up to 15 h after virus acquisition. Seed transmission is usually slight (0-3 %) but instances of 21.5% have been recorded in cowpea cv. Kurodane 16 for a virus which resembles the type strain and 14.7% for a virus which may be related, adzuki bean mosaic (Tsuchizaki et al 1970). Seed transmission ranged from 3 to 19% in cv. Pusa Phalguni for an Indian isolate serologically related to the African (neo-type) virus (Tsuchizak et al 1970). Plants infected through the seed show bright mosaic symptoms in primary leaves. 2.3 Management of CABMV Diseases of Cowpea Different control methods techniques have been employed to reduce CABMV disease incidence on cultivated cowpea. Remove any disease seedling that show symptoms of the disease and place them in a refuse pile away from cowpea production field (Thottapilly and Rossel, 1992). Planting dates can be used as an effective tool for disease management by alternating the planting ages and planting dates for the particular variety grown in order to denaturalize inoculums transfer by vectors (Thottapilly and Rossel, 1992). Scouting and monitoring fields for aphids and disease symptoms regularly can be carried out (Adegbite, 2008). Control methods should not be limited

15 to the use of cultivars with resistance to diseases, pathogen-free seeds, sterilized equipment for transplant production, rouging, eradication of diseased plants and alternate hosts, but also i experimenting with environmental variables such as time of planting, (Adegbite, 2008). Early weed control, application of registered insecticides at least regularly at recommended dosage may reduce aphids and the feeding damage they cause. Control of viruses by insects can be reduced by the destruction of old infected crops well before planting subsequent crops beside alongside them, (Thottapilly and Rossel, 1992).

16 (Agriculture, Abeokuta (UNAAB), Alabata, Ogun state.

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18 After inoculation, the plants were left to pod. The pods were later harvested and the seeds were removed from the pod. Three seeds of each cowpea variety harvested were re- planted in a 5kg sterilized soil. After fully developed, the leaves showing symptoms were randomly selected in each variety and taken to the bio-technology laboratory for serological assay using ACP-ELISA to confirm the presence of CABMV. 3.4 Virus detection Antigen coated plate ELISA (ACP-ELISA) [KPL Technical Guide for ELISA Protocols (on line 1999/2000) and IIT A virology laboratory modified protocols] ACP-ELISA was also used for the detection of the presence CABMV, in all the varieties. Anti-bodies such as: PBS- T (PH 9 Cone. xlo), Goat anti-rabbit alkaline phosphatase conjugate, P-nitrophenyl substrate, Conjugate buffer, fat skimmed milk, coating buffer (coating buffer 2% WW PVP) were collected from the virology laboratory of the International Institute of Tropical Agriculture (IITA) Ibadan Virus Indexing Protocol 100 ml of antigen was added to coating buffer (coating buffer 2% WW PVP). Then, plate was covered and incubated overnight. The following day, the incubated plates were washed three times with PBS- T by flooding three minute each time, drained and tap plate dried. The plates were blocked with 200ml per well of 3% (w/v) dried non-fat skimmed milk in PBS-T. The plates were then covered and incubated at 37 C for two hours. After incubation, ELISA plate were emptied and tap plate dried then 100ml per well of antibody (monoclonal or polyclonal) was diluted in conjugate buffer 1:5000. The ELISA plate were covered and incubated at 37 C for 2 hours. After incubation, plates were washed three times with PBS- T by flooding for three minutes each time and were later tap plate dried. A quantity of goat anti-rabbit alkaline phosphatase conjugate 100ml was diluted in conjugate buffer 1:5000. The 10

19 ELISA plate were covered and incubated for two hours, following this, the ELISA plate were washed three minutes each time and tap plate dried. 200ml per well of O.Olg of p-nitrophenyl phosphate substrate was added to 10ml substrate buffer. For all incubations, plates were covered with ELISA cover plates to avoid edge effect and to maintain uniform temperature. Healthy cowpea seeds were used as negative control while CABMV infected cowpea were used as positive control. After one hour, the absorbence was measured at 405 nm using multiscan ELISA reader. The samples were considered positive when the ELISA reading exceeded that of the healthy control by or was at least twice the reading for the healthy control. 3.5 Statistical Analysis Data collected were subjected to analysis of variance using the Statistical Analysis System (SAS, version 8). Significant means were separated using Least Significant Difference.

20 CHAPTER FOUR RESULTS CABMV was detected in the leaves of ten out of all the fifteen varieties of seeds (IT90K-227-2, TVX 3236, IFE 98-12, IFE 98-14, ERUSU, MODUPE, ZOBO, OLOYIN, DRUM, MALLAM, SOKOTO) analyzed serologically with ACP-ELISA, but was not present in the leaves of the remaining five varieties(ife BROWN, IT , IFE BPC, BROWN BERET and IFE 98-12). The explanation for the five varieties not been infected could be confirmed by this experiment. There were significant differences in the infection rate in all the varieties at P< 0.05 as shown in Table 1.

21 Plate 1: Healthy plant showing no symptom expression of Cowpea Aphid-Borne Mosaic Virus

22 Plate 2: Infected cowpea plant showing Cowpea Aphid-Borne Mosaic Virus symptom expression of chlorosis, necrosis and stunting.

23 Table 1: Mean number of plants mechanically inoculated with CARMV showing symptoms OLOYIN 4.33 MODUPE 6.67 IT90K IFE BROWN 0.33 ERUSU 7.67 IT IFE TVX BROWN BERRET 0.00 IFE BPe 0.00 MALLAM 5.67 ZOBO 6.00 SOKOTO 8.67 DRUM IFE LSD 1.79

24 CHAPTER FIVE DISCUSSION Viruses transmitted through seed have been reported to have certain common characteristics (Johansen et ai, 1994) such as mechanical transmissibility, narrow host range among plants, and vector transmission in a non persistent manner, which also are exhibited by CABMV. The result of this study demonstrate the potential role of seed transmission in the spread of CABMV, the high rate of seed transmission of CABMV observed in this study may be responsible for the wide spread occurrence of the virus in Nigeria. Varieties (IFE BROWN, IT , IFE BPC, BROWN BERET and IFE 98-12) that were not infected may probably be the exhibition of resistant trait to the virus but this could not be ascertained through this experiment. 5.1 CONCLUSION There is need to do a good seed health test before been marketed to farmers, because all the varieties sourced from local market tested positive to the virus. Also, resistant varieties need to be produced to reduce the spread of the virus because it is seed borne virus disease. 5.2 RECOMMENDATION Having discovered in this experiment that cowpea seeds are susceptible to viral diseases, 1 recommend that cowpea seeds should undergo seed health test before they are sold to farmers on large scale.

25 Adegbite, A.A. and Amusa, N.A The major economic field diseases of cowpea in the humid agro ecologies of South- Western Nigeria. Ajr-icanjournal (~l Biotechnology 7: Akinjogunla OJ (2005). Effects of single and mixed inoculation with viruses on symptomatology, growth, yield and nutritive content ofcowpea: (Vigna unquiculata) M.Sc. Thesis. University of Lagos,Nigeria. Bashir M, Hampton RO (1996). Detection and identification of seedborne viruses from cowpea (Vigna unquiculata (L.) walp) germplasm. Plant PathoI. 45: Bock KR (1973). African strains of cowpea aphid-borne mosaic viruses Ann. AppI. BioI. 74: Bock KR, Conti M (1974). Cowpea aphid-borne mosaic virus CMI/AAB. Description of plant viruses No Kew, Surrey,England. Coetzee JJ (1995). Cowpea: A traditional crop in Africa. AtTica crops information 1995: vegetable and ornamental plant institute and the grain crops institutes, agricultural research council. Duke JA (1981). Handbook of Legumes of World Economic Importance. Plenum press, New York pp Fajinmi, A. A., 1995: Studies on pepper veinal mttle Poty virus disease of pepper (Capsicum species) in Ibadan and Environ. M.Sc. thesis, Faculty of science, Department of Botany and Microbiology. University of Ibadan, Oyo state, Nigeria. Pg 65.

26 Hughes JDA, Shoyinka SA (2003). Overview of viruses oflegumes other than groundnut in Africa in Plant virology in sub-saharan African, Proceeding of Plant Johansen, E., Edwards, M. c., and Hampton, R. O Seed transmission of viruses: Current perspectives. Annu. Rev. Phytopathol. 32: Ladipo L Seed Transmission of Cowpea aphid borne mosaic virus in some cowpea cultivars. Nigerian Journal of Plant Protection 3: 3-10.' Rachie KG (1985). Introduction of cowpea research. production and utilization. edicted by singh SR, Rachie John Wiley & sons, Chichester UK. Shoyinka SA (1974). Status of viral diseases of cowpea in Nigeria, in proceedings of the first lit A grain legume improvement workshop. Summerfield RJ, Huxley PA, Steele NN (1974). Cowpea (Vigna unquicu/ata (L) walp.) Field Crop Abstr. 27: Taiwo MA (2003). Viruses infecting legumes in Nigeria: case history. In plant virology sub- Sarahan Africa. Proceedings of Plants Virology. Taiwo MA, Akinjogunla OJ (2006). Cowpea Viruses: Quantitative and qualitative effect of single and mixed viral infection. Afr. 1. Biotechnol. 5(19): Thottapilly G, Rossel HW (1992). Virus of cowpea in tropical Africa. Trop. Pest Manage. 38(4): Tsuchizaki Yora and Asuyam Ann. phytopath. Soc. Japan, 1970a

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