TRANSMISSION OF COWPEA APHID BORNE MOSAIC VIRUS ...

TRANSMISSION OF COWPEA APHID BORNE MOSAIC
VIRUS (CABMV) (genus potyvirus, family potyviridae) IN THE
SEED OF COWPEA
(Vigna unquiculata L. Walp) PLANTS.
BY
BANKOLE, IDRIS ADEBISI
MATRIC NO: 2004/0714
DEPARTMENT
OF CROP PROTECTION
COLLEGE
OF PLANT SCIENCE AND CROP PRODUCTION
UNIVERSITY OF AGRICULTURE, ABEOKUTA, OGUN STATE,
NIGERIA.
A PROJECT SUBMITTED IN PARTIAL FULFILLMENT OF THE AWARD OF
BACHELOR OF AGRICULTURE, (B. AGRIC.) OF THE UNIVERSITY OF
AGRICULTURE,
ABEOKUTA, OGUN STATE, NIGERIA.

.....'1.f.~r?.lll.l.
Date
.t:J.7..1..I~ l~ ("
Date

I dedicate this project to Almighty ALLAH, who through Ramah guard and guided me
through my entire stay in UNAAB. I also dedicate it to the memory of my beloved Dad, Alh.
A.A Bankole whom there can never be any replacement for, and to my dear Mum, Alhaja
.A.O Bankole whom asides ALLAH has been the pillar behind my pole.

All praises and adoration is due to Almighty Allah (S.W.T), the Giver and Sustainer, Who
through His infinite mercy had made this project an easy task for that I am sincerely
grateful.
I want to appreciate the effort of my supervisor Dr. A.A. Fajimi, who through his
constructive criticism made this project an easy one for me, I pray Allah (S.W.T.) make your
entire task an easy one for you. I will like to thank my Head of Department,
Dr. O.R. Pitan
and all other lecturers in the department; you've all made a great impact in my life.
My unquantifiable thanks goes to my channels to this world, firstly my Late father, Alhaji
A.A. Bankole who did everything possible to save us from the hardship of this world and
hereafter, Dad you are second to none and I'll forever miss you, also to my Mum (Iya Idris),
who despite all odds still manage to see us all through our various ambition at every stage of
our lives, I pray you live to do more and abundantly
rip the fruit of your labour. To all my
siblings I am saying a very big thanks to you all, no need to mention your names because
there can never be anyone in this world that will share those precious name with you in my
heart, thanks for the support, I sincerely appreciate it.
My special thanks goes to the Imam of this University Dr. LA. Ayinde who made me move
on at those periods when I thought I could not do it again, I don't know how much to
appreciate your effort on me, thank you very much. To all associations I belonged to
especially M.S.S.N and Muslim Contact Forum (M.C.F.) thank you for adding to my spiritual
well-being.
Lastly to all my friends who had made an impact such as Hammed Adebayo (you remain the
best), Matanmi Ibrahim, Muideen Raheem, Onibon Kazeem, Damola Agboola, Tunji

,
~.,
iAdeY

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Cowpea (Vigna unquiculata L. Walp) is an economically important leguminous crop in
r
Nigeria and several other countries of the world. Among the disease that affects cowpea,

Certification
Dedication
Acknowledgement
Abstract
Table of Content
List of Table
List of plates
CHAPTER ONE
INTRODUCTION
1.1 Cowpea Virus Diseases
1.1.1 Economic Importance of Viral Diseases of Cowpea
1.2 Objective of the study
CHAPTER TWO 4
2.0 LITERATURE REVIEW 4
2.1 Diseases of Cowpea 4
2.1.1 Incidence of viral diseases on cultivated cowpea 4
2.1.3 Cowpea aphid borne mosaic virus (CABMV) 5
2.2 Spread of viral Diseases on Cultivated Cowpea 5
2.3 Management ofCABMV Diseases of Cowpea 6

3.0 MATERIALS AND METHOD 7
3.1 Experimental site 7
3.2 Materials used 7
3.3 Mechanical inoculation 7
3.3.1 Procedure for mechanical inoculation 8
3.4 Procedure for the separation of seed coat from the cotyledon 8
3.4.1 Seed coat and cotyledon test 9
3.5 Serological assay procedure using ACP-ELISA 9
3.5.1 Virus detection 9
3.5.2 Virus Indexing Protocol 10
CHAPTER FOUR
4.0 RESULTS
CHAPTER FIVE
5.0 DISCUSSION
5.1 CONCLUSION
5.2 RECOMMEDATION
REFERENCES

TABLE 1: Detection of CABMV by ACP-ELISA in the cotyledon, seed coat and leaves of
I';
grown out seedlings in ten varieties of cowpea 15

Cowpea (Vigna unquiculata L. Walp) is an important leguminous crop in tropical and subtropical
areas of Asia, Africa and Latin America, as well as parts of southern Europe and the USA (Singh et
al., 1997; Boukar et al., 2004). It was estimated that 3.3 million tonnes of cowpea dry grains were
produced world-wide in year 2000. Nigeria produced 2.1 million tonnes of this, making it the
world's largest producer, followed by Niger (650,000 tonnes) and Mali (110,000 tonnes) (IITA,
2004; Adegbite et el, 2008). According to FAO, about 7.56 million tonnes of cowpea are produced
worldwide annually on about 12.76 million hectares. Sub-Saharan Africa accounts for about 70% of
total world production (lIT A, 2009). Cowpea also provides a significant dietary protein of the people
and plays an important role in the nutritional role in developing countries of the tropics, subtropics,
especially sub-Saharan Africa ( Rachie, 1985, singh et el.,). It provides high protein and lysine
contents make cowpea a natural supplement to staple diets of cereals, roots and tubers commonly
grown in many poor countries (Bressani, 1985; Adekola et el, 2007). Cowpea young leaves and pod
contains vitamins and minerals which have fuelled it's usage for human consumption and animal
feeding.( Rachie, et el., 1985;Nelson,1997). Cowpea is a common food crop throughout Nigeria but
particularly in the middle belt and drier northern regions also in Nigeria cowpea is used to make
soups and bean mixes such as moi- moi and beans cake. The leaves may be boiled, drained, sun
dried, and then stored for later use. (Duke 1981) All of these factors make cowpea a vital crop to
millions of poor people. Cowpea is susceptible to a number of fungal, bacterial, and viral diseases.
Of more than twenty viruses reported on cowpea from different areas of the world, eight are known
to occur in cowpea in Africa (Mali and Thottappilly, 1986; Brunt et al., 1990). Passage of viral
inoculum from diseased plants to their offspring was long thought to be a rare phenomenon. Today,
seed transmission is known to occur for about one-seventh of the known viruses in one or more of

their hosts (Hull, 2002), and the number is increasing. Three important effects of seed transmission
are: (i) direct and/or indirect injury, as even a low incidence of infected seeds sown results in
numerous randomly scattered foci of inoculum, facilitating early secondary spread in the crop
vectors, (ii) survival of viral inoculum
from one crop season to the next; and (iii) several viruses and
viroids have been, and undoubtedly still are, disseminated worldwide through exchange of seeds
having undetected infection (Albrechtsen, 2006).
1.1 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;
Cowpeagolden mosaic virus (CPGMV),
genus Bigeminivirus; Southern bean mosaic virus (SBMV),
genus Sobemovirus; Sunhemp mosaic virus (SHMV), genus Tobamovirus; 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 (Mali and Thottapilly, 1986 ).
Transmission of plant viruses could either be persistent or non-persistent manner (Lana
andAdegbola; 1997). 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,
(Lana andAdegbola; 1997).

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).
1.1.1 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 .Infection by several viruses occurs in cowpea
production which reduces its yield and quality of pod. (Lovisolo et el., 1966)
1.2 Objective of the study
To investigate seed transmission of cowpea aphid-borne mosaic virus (CABMV) in seed coat and
cotyledon of cowpea.

CHAPTER TWO
2.0 LITERATURE 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. (Aboul-Ata et al 1982). Diseased cowpea plants show variable amounts of
dark green vein banding or interveinal chlorosis, leaf distortion, blistering and stunting (Van
Kammen, 1973). Viruses that may be related cause mosaic diseases of adzuki bean (Phaseolus
angu/aris) and asparagus bean (Vsesquipedalis). (Adegbite, and Amusa, 2008).
2.1.1 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
(Brunt,et el 1990).
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 virus detected, (Shoyinka et aI., 1997).
2.1.2 Symptoms of viral diseases of cowpea
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). Typical symptoms of viral infections are stunting, mottling or foliage and
deformed pod.

2.1.3 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 aI1974).
It can cause a yield loss of 13 - 87% under field conditions depending upon crop susceptibility, virus
strain and the environmental conditions, (Bashir,et el,1996). CABMV has spread world-wide
through the exchange of virus-infected germplasm material. The virus-infected seed provides the
initial inoculums 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).
2.2 Spread of viral Diseases on Cultivated Cowpea
Viruses can be transmitted by insects, primarily Aphids, leafhoppers, and thrips, sometimes
whiteflies can transmit viruses. Mites, fungi and nematodes can occasionally transmit viruses (Bock
et el 1974). However, there are two types of viral transmission which could be persistent or nonpersistent.
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 (Vidano, 1965). Non-feeding aphids can retain infectivity up to 15 h after virus acquisition.
Seed transmission is usually slight (0-3 %) but instances of21.5% 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 (Conti et el 1974). Control methods
should not be limited 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 experimenting with
environmental variables such as time of planting. (Crastree et a/1990.)
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)

ț
['
r Ten varieties of Cowpea namely: (IT90K-227-2, TVX 3236, IFE 98-12, ERUSU, MODUPE, ZOBO,

{3A.
~
~3.IJ. Procedure of mechanical inoculation
"easy access of virus into the leaf cells. 600 meshes carborandum
E
powder dusted on each marked leaf.
[. sterilized swab was dipped into the CABMV sap extract gotten from the ground CABMV infected
~pt;and each dusted leaf was stroked from the petiole to the leaf tip with one hand while the other
f band supported the leaf. Five strokes of the CABMV saturated with cotton swab were made on each
~,marked leaf and the inoculums in the pad were renewed frequently. Heavy pressure was avoided and
the same area was not gone over twice.

7 The plates were emptied and tap plate dried then 100ml per well of antibody (monoclonal or
'polyclonal) was diluted in conjugate buffer 1:5000. The plates were covered and incubated at 37°C
f
: O.Olgofp-nitro phenyl phosphate substrate was added to 10ml of substrate buffer.
~" For all incubations,
~
plates were covered with ELISA cover plates to avoid edge effect and to
f.

f biotechnological laboratory in a zip bag for serological assay using ACP-ELISA, to confirm the
~
f: presence of CABMV.
/. 3. Grown out test
t.
~.The third experiment involved plating out of two seeds of each variety of the cowpea used in a
R'
r sterilized covered Petri-dishes during it's early period of growth in three replicates inside the tissue

CHAPTER FOUR
RESULT
In the first experiment (Seed coat and cotyledon assay), CABMV was detected in the cotyledons in
'all the ten varieties of seeds analyzed serologically with ACP-ELISA. In contrast, the virus could not
,be detected in the seed coat of all the ten varieties of seeds analyzed.
While in the second experiment
(Tissue culture), all the leaves that were harvested from the cotyledons of the cowpea varieties that
.'were cultured in a Murashige and Skoog basal medium all tested positive to CABMV when analyzed
logically. Similarly, in the third experiment (Grown out test), all the leaves that were harvested
from the seeds germinated on the sterilized moistened filter paper in a sterilized Petri-dish all tested
,positive to CABMV when they were analyzed serologically with ACP-ELISA. This helped in
confirming that the virus was associated with the cotyledon of the cowpea seed.

TABLE 1: DETECTION OF CABMV BY ACP - ELISA IN THE COTYLEDON, SEED COAT
AND LEAVES OF GROWN OUT SEEDLINGS IN TEN VARIETIES OF COWPEA
VARIETY COTYLEDON SEEDCOAT GROWN OUT SEEDLINGS
2 3 2 3 2 3
OLOYIN + + + + + +
MODUPE + + + + + +
IT 90K-227-2 + + + + + +
ERUSU + + + + + +
IFE 98-14 + + + + + +
TVX 328 + + + + + +
MALLAM + + + + + +
ZOBO + + + + + +
SOKOTO + + + + + +
DRUM + + + + + +
KEY
+= INFECTED
- = UNINFECTED

"transmission in a non-persistent manner, which was also exhibited by CABMV. The frequency of
J
r seed transmission for plant viruses varies from 0 to 100% and rarely exceeds 50%for most of the
~.
~
i viruses.
f

it had been confirmed in this experiment that CABMV is seed borne and can be transmitted
seed farmers are thereby advised to screen seeds before sowing them so as to obtain a virus
teed to reduce the impact of the CABMV.

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