FACULTE DES SCIENCES DE L'AGRICULTURE
ET DE L'ALIMENTATION
DEPARTEMENT DE PHYTOLOGIE
THESE
PRESENTEE
A L1ECOLE DES GRADUES
DE L'UNIVERSITE LAVAL
POUR L'OBTENTION
DU GRADE DE PHILOSOPHIAE DOCTOR (Ph.D.)
PAR
. ' ;
.' !
MAWUENA YAWOVI GUMEDZOE
...... '.'
. /
, "
... ~,. '
........-. __ / , \\\\\\.
MAITRE ES SCIENCES
" 1 ~- \\.':
.
DE L'UNIVERSITE LAVAL
STUDIES OF VARIABILITY OF THE COWPEA APHID-BORNE
MOSAIC VIRUS (CAbMV) COMPLEX IN NIGERIA
JUIN 1985
ACKNOWLEOGEMENT
1 acknowledge the inestimable help of Dr. H.W. Rossel
(International Institute of Tropical Agriculture, Virology Unit) and
Professor A. Asselin (Département de phytologie, Faculté des sciences
de l'agriculture et de l'alimentation, Université Laval) for their
advice, criticisms and financial support throughout the period of this
work and in the preparation of the manuscript.
1 would like to express my sincere gratitude to Dr. G.
Thottappilly (liTA, Virology Unit) for valuable suggestions, comments
and criticisms.
My thanks are due to Ors. W.H. Reeves, E.P. Terry and A.P.
Uriyo of the International Programs and Training for granting me a
research fellowship at liTA (Ibadan, Nigeria) where this study was
conducted.
1 express also my gratitude to aIl the officiaIs of the
same institute.
1 gratefully acknowledge the financial support of
the International Oevelopment Research Centre (IORC, Ottawa, Canada)
throughout the duration of this study.
My grateful appreciation is due to the officiaIs of the
'Université du Bénin' ~omé, Togo) for granting me leave during this
study.
am deeply indebted to Ors. S.A. Tolin (Virginia Polytechnic
Institute and State University, Blacksburg, U.S.A.), S.A. Shoyinka and
B.B. Singh (lITA, Grain Legume Improvement Program) and N.Q. Ng (liTA,
Genetic Resources Unit) for their help, encouragement and advice.
i i
1 would like to thank Ors. 1.0. Akobundu, G.F. Wilson and
Mr. C.W. Agyakwa (liTA, Farming Systems Program) for providing me seeds
of some of the host plant species tested during this work.
Very special
thanks are due to Ors. M.A. Taiwo (University of Lagos, Nigeria) and
F.J. Morales (ICentro Internacional de Agricultura Tropical 1) (ClAT)
(Cali, Colombia) for the supply of antisera.
1 appreciate especially the technical help of the following
persons:
Miss F.B. Ajala, Miss P. Mukolu, Mr. G.G. Adebayo, Mr. Akinlabi
and Mr. F. Ayinde (liTA, Virology Unit).
1 would like to express my
gratitude to Mrs Odette OesBiens (Electron microscopist at Laval Univer-
sity) for taking electron microscopy photographs.
1 thank Mrs. B. Balogun and Mrs. C. Oevin-Ouellette for
their patience and perseverance in typing the manuscript.
To ail my colleagues and friends at liTA (Ibadan), at Laval
University (Canada) and at IEcole supérieure dlagronomie' (Lomé, Togo)
who have given me encouragement, advice and help, 1 am sincerely grate-
fui.
1 further wish toextend my gratitude to the families of
Mr. E.F. Oeganus and Dr. T.L. Lawson for their friendship, care and
concern during my stay at Ibadan.
Finally, the prayers and encouragement of my wife,
my
parents, brothers, sisters and the whole Gumedzoe family are greatly
acknowledged.
CERTIFICATION
This is to certify that this research was carried out by M.Y. GUMEDZOE
of the IDépartement de Phytologie, Faculte' des Sciences de llAgriculture et
de l'Alimentation, Universite Laval, Quebec, Canada: at the International
Institute of Tropical Agriculture (lITA, Ibadan, Nigeria).
~~h
t:pervisor
Supervisor
H.W. Rossel
A. Asselin
Virologist, lITA
Professor
Ibadan, Nigeria.
Departement de Phytologie
(FSAA) Université Laval
Québec, Canada.
l -6haii.. not cU.e.,
But Uve.,
And de.c1.aJte.
The. WOJLk..6 06 the. LOM.
(Ps.118.17)
AB5TRACT
Both farmers ' fields and experimental plots in Nigeria's
cowpea (Vigna unguiauZata) producing areas were surveyed for ~lants
showing symptoms of virus infection (mosaic t veinbanding).
Cowpea aphid-
borne mosaic virus (CAbMV) isolates were identified from various ecolo-
gical areas of the country.
This potyvirus was recovered from Bambarra
groundnut (Vigna subterranea) t African yam bean (SphenostyZis stenoaarpa)
and wild cowpea (Vigna unguiauZata ssp. dekindtiana).
The identification
was based on reactions of isolates on selected cowpea cultivars t host range
and serological studies as weIl as electron microscopic examination.
Eight CAbMV isolates were partially characterized by their
biological and serological properties.
They differed in severity of
symptoms in cowpea cultivars and many other leguminous plants.
But
their virulence in one plant species did not necessarily paraI leI that
in other species.
The CAbMV isolates exhibited considerable variability
in virulence. Eight virus isolates could be classified into three groups.
Members of the first group were lAT-S' tilT 15 1 and 'IT 16 1 which
produced bright yellow mosaic on susceptible cowpea cultivars and
could not infect Niaotiana benthamiana.
The second group with 'BT-q't
'ONNE ' and IWC' induced mosaic with variable dark green and yellow pat-
ches.
The isolate 'BT-q' was the most severe and virulent one.
The
third group of isolates included
'IT 11'
and
'Zaria'
which
produced mild green mosaic and veinbanding symptoms on different cowpea
lines.
N. benthamiana was infected by members of the second and third
groups of isolates.
V. subterranea was found to be a new susceptible
host plant species for our CAbMV isolates.
vi
Variability in virulence of isolates has important implica-
tions for the Grain Legume Improvement Program at liTA.
Screening for
resistance to CAbMV should be done with representative types of this
virus.
Isolates 'BT-4', 'ONNE ' , liT 111 and liT 16 1 could be used for
screening studies.
An evaluation of cowpea germplasm accessions and
breeding lines for their resistance to our isolates has shown that
germplasm accessiorts TVu 401 and TVu 1948 possess high levels of resis-
tance to
aIl CAbMV isolates.
The advanced breeding lines IT 820-752,
IT 820-885, IT 820-889 and IT 82E-60 were found to be resistant to at
least 6 of CAbMV isolates.
Rates of seed transmission of four of the CAbMV isolates
were low and variable with cowpea cultivars.
Consequently, efforts must
be directed towards the production of virus-free seeds for international
transfers of improved cowpea
varieties.
A selection for resistant cow-
pea lines to locally-occurring CAbMV isolates should help to reduce the
incidence of this virus where cowpea
is
intensively grown.
Symptom-
less cowpea lines transmitted virus through seeds.
These results support
the view that it is more useful to breed cowpeas for resistance to infec-
tion with CAbMV than for tolerance.
Antisera prepared against two of our isolates ('ONNE ' and
'Zaria ' ) and two other isolates of the same virus (Kenya and Nigeria
isolates) reacted positively with aIl CAbMV isolates.
However, intensity
of the reactions varied with isolates.
AlI isolates belong to the same
serological group.
Previous reports on potyvirus(es) in cowpeas in Nigeria
were essentially dealing with CAbMV.
Results obtained during our
studies show sorne evidence that blackeye cowpea mosaic virus (B1CMV)
could occur in that country.
Both viruses were identified as 'CAbMV
complex ' .
RESUME
Le n i ébé ou 1cowpea' (Tfigna unguiau Zata (L.) Wa 1p.) es tune
légumineuse très importante pour les pays africainset plus particulière-
ment le Nigéria qui est au premier rang de la production mondiale.
En
effet, clest une plante exploitée à différentes fins (al imentation hu-
maine, plante fourragère, engrais vert, lutte contre l'érosion, etc.).
Malheureusement, chaque année, plusieurs maladies réduisent
les rendements de cette culture dans des proportions très considérables.
Parmi les maladies graves du niébé figurent de nombreuses viroses.
Une
de ces dernières, la mosaltque du niébé transmise par les pucerons est
causée par un potyvirus: le CAbMV (= Icowpea aphid-borne mosaic virus').
Ce virus a retenu notre attention au cours de ces études.
Différents
isolats de ce virus ont été collectés tant dans les champs des paysans
que dans des parcelles expérimentales de différentes régions productri-
ces du niébé au Nigéria.
Ceux-ci ont été identifiés et caractérisés par
différents méthodes: gamme de plantes différentielles, symptomatologie,
sérologie et microscopie électronique.
CAbMV a été retrouvé infectant
naturellement les plantes suivantes:
Vigna subterPanea~ V. unguiauZata
sp.
unguiauZata~ V. unguiauZata sp. dekindtiana et SphenostyZis steno-
carpa.
Il ressort de ces études que huit des isolats peuvent être
classés en trois groupes suivant le type et la sévérité de leurs symptô-
mes sur différentes 1ignées du niébé.
Le premier groupe comprend les
isolats lAT-5 1 ,
liT 15' et 'IT 16 1 qui induisent une mosa1tque jaune
intense sur les niébés sensibles.
Le deuxième groupe avec les trois
isolats IBT-4', 'ONNE' et 'WC' produisent une mosal(que d'intensité
vii i
variable sur le niébé.
les deux autres isolats' IT 11' et 'Zaria' déve-
loppent une mosaVque verte accompagnée parfois d'un liséré des nervures
('veinbanding ' ).
Niaotiana benthamiana est sensible aux isolats 'BT-4',
'IT 11', 'ONNE', 'WC' et 'Zaria'.
Vigna subterranea est une nouvelle
plante-hôte pour les isolats du CAbMV.
Cette variation de la virulence notée au sein des isolats
du CAbMV a des conséquences importantes pour l'amélioration de cette légu-
mineuse à graines à l'Institut international d'agriculture tropicale
(liTA) à Ibadan (Nigéria).
D'abord, le criblage des lignées ou variétés
sélectionnées du niébé devra se réaliser avec un échantillon d'isolats
de ce virus assez représentatifs.
les isolats 'BT-4', 'ONNE', 'IT 11'
et 'IT 16 1 sont suggérés pour de telles études de criblage.
l'évaluation
de lignées provenant de collections du matériel génétique du niébé à
1Il ITA a permis d'identifier deux lignées résistantes TVu 401 et TVu 1948.
les cultivars améliorés du niébé: IT 820-752, IT 820-885. IT 820-889 et
IT 82E-60 ont été évalués aussi et se sont montrés résistants à au moins
six de nos isolats.
les taux de transmission de quatre des isolats viraux par
la semence sont variables.
Mais ces taux sont suffisamment élevés pour
permettre le développement d'épiphyties graves si les graines infestées
sont utilisées.
Il a été noté aussi que des cultivars tolérants (sans
symptômes) du niébé transmettent aussi le virus par la semence.
Il est
donc plus intéressant d'util iser des variétés résistantes que celles
tolérantes au CAbMV.
Dès lors, les efforts doivent être orientés vers
la production de semences indemnes de virus, surtout pour les lignées
destinées à des échanges internationaux de matériel végétal amélioré.
Ensuite, la sélection et l'utilisation de variétés résistantes du niébé
peuvent permettre de réduire l'incidence de cette virose là où le niébé
est cultivé de façon intensive.
les antisérums produits contre deux de nos isolats ('ONNE'
et 'Zaria') et deux autres souches du même virus ('Kenya' et 'Nigeria')
réagissent positivement avec tous les isolats du CAbMV par la méthode
sérologique de double diffusion dans l'agar.
Ces résultats indiquent
que nos isolats appartiennent tous au même groupe sérologique.
ix
Jusqu'à présent. seul le CAbMV a été rapporté du Nigéria,
mais nos résultats semblent indiquer qu'un autre potyvirus. le BICMV
(= 'Blackeye cowpea mosaic virus') proche du CAbMV. serait aussi présent
dans ce pays. Les deux virus ont été désignés ici sous le nom générique
du 'complexe CAbMV'.
TABLE OF CONTENTS
Page
ACKNOWLEDGEHENT •••.••••.•••.•••.•••••••••••••••••••••••••••••••
CE RT 1FI CATION ••••••••••••••••••••••••••••••••••••••••••••••••••
i i i
DEDI CATION.....................................................
iv
ABSTRACT
......................................................
v
RE SUME •••••••••••••••••••••••••••••••••••••••••••••••••••••••••
vii
TABLE OF CONTENTS ••••••••••••••••••••••••••••••••••••••••••••••
x
LIST OF TABLES.................................................
xiv
LIST OF FIGURES
••••••••••••••••••••••••••••••••••••••••••••••••
xvi
LIST OF ABBREVIATIONS
•••••••••••••••••••••••••••••••••••••••••• XVIII
1NTRODUCT ION ••••••••••••••••••.•••••••.••••••••••••••••..•.••••
CHAPTER 1 -
LITERATURE SURVEY .................................
5
1.1
Vigna unguiaulata (L,~) .Walp •..•...••••.•..•..••...
5
. ,.
1. 1. 1
Taxonomy .: .',~-/.,/~~~ ...• ~.~ .'.~ •....•.•••.••....•
5
1. 1. 2
Origin
{,';'./.. ~.F'~ I.e ••••',
.
:-:
5
~
',,- '.-~- ,'.
.
1. 1. 3
Distributi~ land ut1-)'iza/t[op •••••••••••••••
.
6
,.,~
,
.' '-'
1. 1.4
Prod~ct 1on .. -. ~ • ',~~~.:-.:;.r' ' y~
.
8
•
~~l.
. :,.,.
1. 1. 5
Major cowpea
vlrus,es"j,t"'Nigeria
..
8
1.1.5.1
Cowpea (yellow) mosaic virus
(CYHV)
••••••.•..•.•••.•...•..•....
la
1.1.5.2
Cucumber mosaic virus:.
cowpea strain (CuMV)
11
xi
Page
1.1.5.3
Cowpea mott1e virus (CMeV) .....•..
11
1.1.5.4
Cowpea golden mosaic virus
(CGMV) •.•..............•.....•....
12
1.1.5.5
Southern bean mosaic virus:
cowpea strain (SBMV-CS)
.
12
1.1.5.6
Cowpea mi1d mott1e virus
(CMMV) ........•••..•.........••...
13
1.1.5.7
Tobacco mosaic virus: cowpea
stra i n (TMV-CS) ........•..........
13
1.2
Viruses of the cowpea aphid-borne mosaic
virus (CAbMV) comp1ex .....•..•...•........•..•••.•
14
1 . 2. 1
Geographica1 distribution and economic
importance of CAbMV and BI CMV .•.•..•..••..•
14
1.2.2
Bio1ogical characteristics .....•........•..
16
1.2.2.1
Sources of inoculum •.....•...•.•..
16
1.2.2.2
Transmission through seeds
and by vectors ........•..••..••...
16
1.2.2.3
Host range and symptomatology
17
1.2.3
Strains and/or isolates described
in the literature •.........•.........•••...
26
1.2.4
Separation of viruses from mixed
infections (combinat ions of CAbMV,
BICMV and CuMV or other cowpea viruses)
26
1.2.4.1
Inoculation of differential
test plants ..•.......•..•..••...••
27
1.2.4.2
DifferentiaI transmission
by vectors •.•....•.•..•••.....•...
28
1.2.4.3
SeriaI transmission by a
vector •.•.•...•••.•••.•....•......
28
1.2.4.4
Seed transmission .........••......
28
1.2.5
Identification of sources of
res i stance ..•.•..•....•.....•.•......•....•
28
1.2.5.1
The eva1uation of germplasm
access ions ..•.•.•..••..•.........•
29
1.2.5.2
The evaluation of advanced
b reed i ng 1i nes ..........•......•..
29
1.2.6
Phys i co-chem i ca 1 p rope rt ies .••.•.•.••.••..•
30
1.2.6.1
Pur i f i ca t i on ••....••...........•••
30
1.2.6.2
Morphology and partic1e sizes •...•
31
1. 2.7
Antigenic properties of the virus ......•.•.
31
1.2.7.1
Microprecipitin test •...•...•..•..
32
1.2.7.2
SOS - immunodiffusion tests .•..•.•
33
xi i
Page
1.2.7.3
Enzyme-l inked immunosorbent
assay (ELISA) ..
33
1.2.7.4
Immune electron microscopy .......•
34
CHAPTER 2 -
MATERIALS AND METHODS ..............•.............•
35
2.1
General approach
35
2.2
Test plants...................... ..... ••... •.. ....
36
2.3
Survey
36
2.4
Identification of isolates
38
2.4. 1
Electron microscope examination
38
2.4.2
Symptomatology on selected test
plants ..•.....................••...........
38
2.4.3
Prel iminary serological studies ....•.......
39
2.5
Isolates and their maintenance
39
2.6
Artificial host range and symptomatology
40
2.7
Differential reactions on various cowpea
germplasm accessions and breeding lines .....•.....
41
2.8
Transmission studies
41
2.8.1
Aphid transmission ..................•...•..
41
2.8.2
Seed transmission ........•................•
42
Pur i fi ca t ion
.
42
2.10
Ultraviolet absorption spectrum of purified
virus ........•......•............•................
43
2. 11
Se ro log i ca 1 st ud i es
.
43
2.11.1
Antiserum production .............•........
43
2.11.2
SDS - immunodiffusion tests
..
44
CHAPTER 3 -
RESUL TS AND DI SCUSS 1ON
.
46
3. 1
Results
46
3. 1• 1
Su rvey .................................•...
46
3. 1.2
Identification of the isolates
.
47
3.1.3
Host range and symptoms
:
.
51
xii i
Page
3.1.3.1
Local lesion
hosts ...•....•......
51
3.1.3.2
Leguminous species with
systemic reactions
.
52
3.1.3.3
Non-Ieguminous plants with
systemic infection .............•..
53
3.1.3.4
Non-hosts for both viruses
.
57
3.1.4
Reactions of CAbMV and/or B1CMV
isolates on cowpea cultivars, germ-
plasm accessions and breeding lines
59
3.1.4.1
Reactions on cowpea germ-
plasm accessions .......•...•......
59
3.1.4.2
Reactions of isolates 'ONNE',
'IT 11 1 ,
liT 16' and 'Zaria'
on fort y cowpea breeding 1Ines
63
3.1.5
Transmission studies ....................•..
69
3.1.5.1
Aphid transmission ...•......••••..
69
3.1.5.2
Seed transmission of the
isolates 'ONNE', lIT Il',
'Zaria' and 'IT 16' in fort y
cowpea breeding lines ...•.........
69
3.1.6
Purification of the isolates
.
75
3.1.7
SDS-immunodiffusion tests .............•....
75
3.2
Discussion ........................................
76
3.3
Conclusion ........................................
84
LITERATURE CITED .•...•.......•..
.......•.
87
LIST OF TABLES
Page
TABLE 1
Major viruses reported from cowpeas
in Nigeria and sorne of their properties
9
TABLE 2
Geographical distribution of viruses
of the cowpea aphid-borne mosaic virus
complex (B1CMV and CAbMV)
.......•...........••
15
TABLE 3
Selected susceptible plant species to
B1CMV and/or CAbMV .•.........•..•...•.........•
18
TABLE 4
Comparison of physico-chemical properties
of BI CMV and CAbMV .•.....•....••••.......•.•.•.
32
TABLE 5
Origin of the virus isolates ........•..........
40
TABLE 6
Reactions of CAbMV complex isolates to
different antisera in double immuno-
diffusion tests (serological identifi-
ca t ion)
.
49
TABLE 7
Reactions of CAbMV and/or B1CMV isolates
on selected differential cowpea cultivars
50
TABLE 8
Reactions of CAbMV isolates on selected
susceptible plants ............•.•..•.•.....••.•
58
TABLE 9
Reactions of B1CMV isolates on selected
host plant species ••.•.....•..•..•....•.......•
58
TABLE 10
Reactions of CAbMV and B1CMV isolates on
selected cowpea germplasm accessions •.•.....•..
62
TABLE 11
Reactions of cowpea breeding lines and
germplasm accessions to four isolates
of CAbMV and/or B1CMV •........•...•...•.•..•.••
67
xv
Page
TABLE 12
Selected sources of resistance (cowpea
lines) to B1CMV and CAbMV (Nigerian isolates)
70
TABLE 13
Transmission of CAbMV and/or B1CMV
isolates by Aphis craccivora to V.
unguiculata cv. Ife Brown ••.••••.••..•••••••.•.
71
TABLE 14
Results of seed transmission of isolates
1 ONNE l,
'Za ria l, liT 16 1 and lIT 11 1 in
fort y cowpea breeding lines
..
72
TABLE 15
Seed transmission rates in selected cowpea
breeding lines inoculated with isolates
1 ONNE l ,
1 Za ria l,
liT 16 1 and liT 11 1 •••••••••••
74
TABLE 16
Reactions of the B1CMV and/or CAbMV isolates
to homologous and heterologous antisera and
to antisera of BCMV and BYMV in SDS-
immunodiffusion tests ....••.•..•..•..•..••....•
77
LIST OF FIGURES
Page
Figure 1
Map of Nigeria showing the major and
minor producing areas of cowpeas (Vigna
unguiaul,ata (L.) Wa 1p.) ..••............•••..•.
7
Figure 2
Outline of present studies
37
Figure 3
Electron micrographs of virus particles
of CAbMV •..................................•..
48
Fi gu re 4
Necrotic local le~ions induced by iso-
late 'WC I on Phaseol,us vul,garis L.
cv. Black Tu rt 1e ..•....•.•...........•......•.
54
Fi gu re 5
Symptoms of mosaic, leaf deformation and
veinbanding produced by isolate 'BT-4 1 on
Vigna subterranea (Thouars) Verdc. (Vo 7326)
55
Figure 6
Symptoms of mosaic and leaf deformation
(puckering) induced by isolate lAT-5 1 on
Vigna subterranea (Thouars) Verdc. (Vo 7326)
56
Figure 7
Symptoms of bright yellow mosaic (B) and
commen mosaic (A) induced by isolates
IAT-5' (B) and 'BT-4 1 (A) on Vigna ungui-
aul,ata (L.) Walp. cv. Ife Brown .•.............
60
Fi gure 8
Symptom of bright yellow mosaic induced by
isolates 'AT-5' on Vigna unguiaul,ata (L.)
Walp. TVu 645 (A) and liT 151 0n V. unguiau-
Zata cv. 1fe Brown (B) .•.•..••......•.........
61
Figure 9
Symptoms of CAbMV isolates on Vigna ungui-
aul,ata (L.) Walp. TVu 128 (A and B) ••.•...•...
64
Figure 10
Symptoms of CAbMV isolates 'IT 16' (A),
'Zaria' (B) and isolate 'ONNE' (C) 'on the
cowpea breeding line IT 820-709 .•.............
65
xvi i
Page
Figure 11
Symptoms of bright yellow mosaic and
veinbanding induced by CAbMV isolate
liT 16' on the cowpea breeding line
TVx 3236-6-1 ...................•..............
66
LIST OF ABBREVIATIONS
AS:
Ant i serum
BCMV:
Bean common mosaic virus
B1CMV:
Blackeye cowpea mosaic virus
BYMV:
Bean yellow mosaic virus
CAbMV:
Cowpea aphid-borne mosaic virus
CGMV:
Cowpea golden mosaic virus
CMeV:
Cowpea mottle virus
CMMV:
Cowpea mild mottle virus
CsC 1:
Cesium chloride
CszSO 3:
Cesium sulfate
CuMV:
Cucumber mosaic virus-cowpea strain
CYMV:
Cowpea (yellow) mosaic virus
d:
Dalton (molecular weight unit)
DEP:
Dilution end point
ELI SA:
Ensyme-linked immunosorbent assay
FAO:
Food and Agriculture Organization of the United Nations
Ha:
Hectare
liTA:
International Institute of Tropical Agriculture
IT 820-]09:
(and other IT numbers):
are cowpea breeding 1ines from liTA
Kg:
Ki logram
LI V:
Longevity in vitro
mL:
Mi 11 i 1i ter
MV:
Molecular weight
NaCl:
Sod i um ch 10 ride
NAz-EDTA:
Disodium-ethylene diamine tetraacetic acid
NaZS03:
Disodium sulfite
xix
Sodium azide
nm:
Nanometer
00:
Optical density
PB:
Phosphate buffer
PEG
:
Polyethylene glycol-8000 (molecular weights between 8000
8000
and 9000)
PTA:
Phosphotungstic acid
RNA:
Ribonucleic acid
S:
Sedimentation coefficient unit (Svedberg)
SBMV-CS:
Southern bean mosaic virus - cowpea strain
SOS:
Sodium dodecyl sulfate
SMV:
Soybean mosaic virus
SRNA:
Sedimentation coefficient unit (Svedberg) for RNA (SRNA)
SV:
and for virus particles (SV)
TIP:
Thermal inactivation point
TMV-CS:
Tobacco mosaic virus - cowpea strain
TVu:
Tropical Vigna unguiculata (from germplasm collection)
TVx 3236
(and other TVx numbers):
Cowpea crosses obtained at liTA
INTRODUCTION
Cowpea (Vigna unguiaulata (L.) Walp.) is an ancient crop
grown throughout the tropics and sub-tropics.
It is indigenous to tro-
pical Africa from where it was introduced into other tropical and sub-
tropical regions of the world (Cobley and Steele, 1975).
Although the
crop is cultivated in most of the states of Nigeria, it is predominant-
ly grown in the Guinea and Sudan savannah regions of the Northern
Nigeria.
ln fact,Nigeria produced 77.5% of the world's production of
cowpea seeds (FAO, 1975).
Cowpea
has
a high seed-protein content that
has made
it
an important source of protein for human and animal nutri-
tion (Oyenuga, 1968).
The seeds are prepared and consumed in various
forms in Nigeria and in parts of West Africa and India.
In addition,
the holms constitute a major
source of fodder for livestock.
More than 80% of the world's production of cowpea is from
African countries (FAO, 1975).
But the average yield from farmers·
fields in Africa is very low (between 200 and 300 kg/ha) (Summerfield
et al., 1974).
Susceptibility to diseases and insect pests and several
other constraints are responsible of su ch low yields.
Several vi ruses naturally infect cowpea
in Africa, and
these occur often in mixed infections (Thottappilly and Rossel, 1984).
The following are those reported from Nigeria: cowpea aphid-borne mosaic
virus (CAbMV), cowpea (yellow) mosaic virus (CYMV), cowpea mottle virus
(CMeV), cucumber mosaic virus-cowpea strain (CuMV), southern bean mosaic
2
virus (SBMV), cowpea mild mottle virus (CMMV), cowpea golden mosaic
virus (CGMV) and cowpea strain"of tobacco mosaic virus (TMV).
The first two constitute
a
threat to cowpea production
in that country.
Complete loss of a cowpea crop grown in dry season
under irrigation in Northern Nigeria in 1974 was tentatively attributed
to CAbMV (Raheja and Leleji, 1974).
Another potyvirus, closely related
although described distinct from CAbMV is blackeye cowpea mosaic virus
(B1CMV).
It was originally reported from the United States of America
(Anderson, 1955a;
Lima et aZ., 1979;
Zettler and Evans, 1972) but not
previously identified in Nigeria.
Together these two viruses are
referred to as the cowpea aphid-borne mosaic virus complex in this thesis.
CAbMV and/or B1CMV seem to cause the most widespread virus
diseases of cowpea in Nigeria.
They are aphid-and seed-transmitted
viruses and they are capable of reducing considerably the productivity
of this crop as a result of high epidemic potential.
Since the CAbMV complex seems to be an economically very
important constraint on cowpea production in Nigeria, a good understand-
ing of its components as regards to identity and variability is con-
sidered essential to develop effective control measures.
The main
measures for control are the following:
1.
Removal or avoidance of sources of infection, which may
be relevant only in the case of seed-borne infections, and
2.
Improvement in genetic resistance of the crop.
The first measure of control mentioned is particularly
impracticable, mostly inadequate or even impossible particularly in the
case of developing countries.
Therefore, a major, if not the sole solu-
tion, may then be the growing of resistant cultivars, if these are
available.
When this is not the case, studies are needed to identify
sources of resistance and when necessary, these resistances need to be
incorporated into agronomically acceptable and lo~ally preferred geno-
types.
3
ln the case of CAbMV and B1CMV, cowpea cultivars have been
identified which possess resistance to one or both of these two viruses
(Ladipo and Allen, 1979a;
Taiwo, 1978).
Whereas sources of resistance
to CAbMV or B1CMV have been found, certain questions still remain
unanswered.
1.
Do both types (CAbMV and B1CMV) occur in Nigeria 1
2.
What is the natural variability of CAbMV and/or
B1CMV in Nigeria 1
3.
Is it possible to define certain biological or sero-
logical groupings among
the isolates obtained during
our studies 1
4.
What is the relative importance of the various types
identified in Nigeria 1
5.
Is it sufficiently safe in resistance screening studies
to use one particular isolate or a few isolates each
representative of' a particular type 1
6.
Do these isolates represent CAbMV and B1CMV as defined
according to criteria obtained from the literature 1
7.
Is this distinction valid as concluded from findings
obtàined during our studies 1
The answers to these questions may have important 'practical
implications for the cowpea program at the International Institute of
Tropical Agriculture (liTA, Ibadan, Nigeria)
in its efforts to develop
improved varieties which possess resistance to this virus complexe
Since this study was initiated to determine the occurrence
and variability of CAbMV and/or B1CMV and its possible implications for
the cowpea improvement program at liTA, the main objective of the
research activities reported in this thesis was to collect and identify
different types of CAbMV and/or B1CMV and differentiate them on the
basis of biological, serological and physico-chemical properties in
reference to the available literature on these viruses.
4
We expect that the new information obtained with CAbMV and
B1CMV will contribute to a better understanding of the identity and
relationships between the isolates obtained and characterized during
our studies.
This is considered essential for effective control by
means of the growing of resistant cultivars.
It is also expected to
lead to a better understanding of the actual geographical distribution
of these two viruses.
CHAPTER 1
LITERATURE SURVEY
1. 1 Vigna unguiaulata (L.) Wa 1p.
1.1.1
Taxonomy
The term cowpea
is used to denote the botanica1 species
Vigna unguiauZata (L.) Wa1p. and its five sub-species as described by
Verdcourt (1970) whose recommendations seem to have been wide1y accept-
ed.
1t is a member of the fami 1y Papi 1ionaceae, the 1argest of the
three divisions of the Leguminosae.
The cu1tivated sub-species are
unguiauZata, catjang and sesquipedaZis.
We have to mention a1so two
other sub-species mensensis and dekindtiana which are wi1d and weedy
cowpeas of the African forest and savannah zones respective1y (Stee1e
and Mehra, 1980).
In genera1, aIl the sub-species are fu11y inter-
fertile.
1. 1.2
Origin
Because of the 1ack of archaeo1ogica1 evidence, the centre
of origin of cowpeas is uncertain and has been various1y reported as
5
6
possibly Asia (India, China), Africa (Ethiopia, Nigeria). Persia or
even South America (Summersfield et al., 1974).
The large distribution of wild cowpeas in the African
savannah and forest zones, especially in West Africa (Nigeria), indi-
cates that this crop is indigenous to Africa from where it was intro-
duced into other tropical and sub-tropical countries (Cobley and
Steele, 1975;
Rawal, 1975;
Steele and Mehra, 1980).
1.1.3
Distribution and utilization
Because of its multiple utilization, cowpea is now grown in
many parts of the world (Africa, India, South East Asia, The West Indies,
The United States of America, Austral ia and parts of Europe and South
America).
In Nigeria the cultivation of cowpea extends over a wide
range of environments, between 5° and l4°N latitude.
The Northern
part (between 9° and l4°N latitude) of the country is most suitable for
this crop, long-day varieties being grown there
from June to November
(Figure 1).
Day-neutral and short day varieties are grown in the less
sunny and wetter south area.
Cowpeas are often grown as an intercrop rather
than as a sole crop.
They are commonly interplanted with sorghum (Sorghum
bicolor (L.) Moench.), millet (Pennisetum typhoides (Burm. f.) Stapf. &
Hubbard) , cassava (Manihot esculenta Crantz), yam (Dioscorea sp.), maize
(Zea mays L.) and cotton (Gossypium sp.) (Ebong, 1965).
Cowpeas possess a large seed-protein content that has made.
them an important source of protein for human and animal nutrition
(Oyenuga, 1968).
The seeds are prepared and consumed in various forms
in Nigeria and in parts of West Africa and India.
Cowpeas are often
used as fodder crop for 1ivestock in some countries (Prata Neves, 1978).
This plant is also grown as a vegetable crop, either for its leaves
which appears to be particularly popular in East Africa (Martin and
Ruberté, 1975), or for the green pods.
The spreading habit of most
varieties makes them effective in the control of erosion.
14°
Nf
9°1
~
--
KEY
3 TO 5 STARS = MAJOR
5 0 ; ' ~O
OOICM
1 TO 2 STARS = MINOR
4°
Figure 1.
Map of Nigeria showing the major and minor producing areas of cowpeas
(Vigna unguicuZata)
-.....J
8
1.1.4
Production
The total hectarage under cowpea in the wor1d is about
5,170,000 ha.
Out of this 4,500,000 ha are in Nigeria a10ne (FAO, 1975).
The estimated annua1 wor1d production of cowpea seeds is 1,097,000 me-
tric tons and 850,000 metric tons are produced in Nigeria or rough1y
77.5% of the wor1d production (FAO, 1975).
In the North of Nigeria,
some 418,000 tons of cowpea seed are produced annua11y on 2.4 mi11 ions
hectares, near1y a1ways intercropped with cerea1s (Ebong, 1965).
More than 80% of the wor1d production of cowpeas are from
African countries (FAO, 1975).
But the average cowpeas yie1d from
farmers' fields in Africa is probab1y between 100-130 kg/ha (Summer-
field et aL., 1974).
However, under intensive1y managed systems, yie1ds
of 1,000-2,500 kg/ha have been obtained (liTA, 1975). The yie1d potentia1
of cowpea grown as sole crop is over 1,000 kg/ha.
But pests and
diseases, especia11y virus diseases, severe1y 1imit the production
of
cowpea (Rossel et aL, 1985;
Singh and Allen, 1980;
Thottappi11yand
Rossel, 1984).
1.1.5
Major cowpea viruses in Nigeria
A1though many viruses have been reported as infecting cow-
pea in Nigeria, on1y eight of them have been so far accurate1y identi-
fied and described (Table 1).
Among them, six can be considered as
major ones: cowpea aph id-borne mosa i c v i rus (CAbMV) (Lad i po, 1976;
Rosse1,1977);
cowpea (ye110w) mosaic virus (CYMV) (Chant, 1959;
Gi 1mer et aL, 1974;
Wi 11 iams, 1975);
the cowpea strain of cucumber
mosaic virus (CuMV) (liTA, 1978;
Thottapi11y and van Lent, 1983);
cow-
pea mott1e vi rus (CMeV) (Shoyinka et aL, 1978);
the cowpea strain of
southern bean mosaic virus (SBMV-CS) (Ladipo, 1975;
Shoyinka, 1974;
Shoyinka and Okusanya, 1975;
Shoyinka et aL., 1979) and cowpea golden
mosaicvirus (CGMV) (Anno-NyakoetaL, 1983;
liTA, 1978.1979).
Two
other cowpea viruses were occasionna11y reported in Nigeria:
cowpea
mi1d mott1e virus (CMMV) (liTA, 1978) and the cowpe~ strain of tobacco
mosaic virus (TMV-CS) (Chant, 1959;
Lister and Thresh, 1955;
Williams,
1975).
TABLE 1.
"aJor vi ruses reported from eowpeas ln Nigeria and same of thelr propertles
CAb"V
CY"V
CG"V
CHeV
t"KV
Cu"V
SB"V-CS
T"V-CS
GIIOUP
Potyvlrus
Comovlrus
Gemln 1vi rus
1 (ungrouped)
tarlavlrus
Cucumovl rus
Sobemovlrus
ToballlOvlrus
"OIlPHOLOGY
Isometrle
FI 1amen tous
Isometrle
Isometrl e
FII_ntous
Isometrle
Is~trle
IIlgld rod
(Virus partleles)
ln pairs
SIZE
Il JI 750 nm
20 Il 21t nm
-
]0 nm
13 Il 650 nm
28-30 n.
25 OIa
18 Il 300 nm
VECTOIIS
Aphlds
Beet les
Whl teflles
Beetles
Whlteflles
Aphlds
Beet les
1
SAP
+
+
-
+
+
+
+
+
TIIANS"ISSION
SEED
0-3'
1-5%
-
10%
2-90'
"-26t
3-'"
1t-20%
O-Ift
HOST RANGE
Wlde
L1ml ted
L1ml ted
Wlde
Wlde
Wlde
L1.1 ted
Wlde
SYMPTOHS
Hasale
Hasale
Bright
Hasale
Hattllng
Hasale
Haule
Hasale
velnbandlng
yellow
moU Ilng
mott Ilng
patehes
YIELD
LOSSES
15-87'
60-100%
90%
75%
-
l''.n
-
5-25%
KEY liE FE !lEIICES
Aboul-Au . t
Chan t
(1959)
Anno-Nyako et al.
1I0bertson (196])
Brunt and
lITA (1978)
Ladlpo (1975)
Chant. (1959)
al. (982)
Gllmer et al.
( 198])
Shoylnka et al.
Kenten (197")
Robertson (1966)
Shoylnka et al.
Lister and
Lacllpo (1976)
( 1971t)
liTA (1978)
( 1978)
Thottappillyand
(1979)
Thresh (1955)
Rossel (1977)
Williams (1975)
lIosse 1 (198")
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:;
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4,344
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QI
(
;:;,.•., ;
4)[,iL
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"'îi,.~
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,.,
10
Most of these viruses have detrimental effects on cowpea
and constitute major constraints to increased production of this crop.
Some of their biological characteristics (except CAbMV) are as follows.
1.1.5.1
Cowpea (yellow) mosaic virus (CYMV)
Cowpea (yellow) mosaic virus is a serious disease of cowpeas
in Nigeria and yield reductions of 60 to 100% have been reported (Chant,
1960;
Gilmer et a~., 1974;
Shoyinka, 1974).
The economic 1055 depnds
on three factors: the virus isolate, the cowpea cultivars and the age of
susceptible plants at the time of infection (Gilmer et a~., 1974).
Early
infections of cowpea seedl ings by CYMV (seven days after emergence)
reduced yields by 40 to 60%, but late infections (after flowering) caused
reductions of only 10-15% (Gilmer et aL, 1974).
CYMV naturally infected
other leguminous plants: soybean (G~ycine max (L.) Merr.), Jack bean
(canava~ia ensifo~is (L.) D.C.), African yam bean (Sphenosty~is steno-
carpa (Hochst ex. Rich.) Harms) and lima bean (Phaseo~us ~unatus L.)
(Gilmer et a~., 1974).
Experimental host range studies showed that
CYMV infected several legume and non-legume species.
Symptoms induced
by CYMV ranged from mild to intense yellow mosaic with blistering of
•. -_o.
leaflets, leaf distort ion end mottl1ng (Chant, 1959;
Gi lmer et aL,
1974).
The vi rus was tr'~~smitt~~ t'hr~\\Jgh seeds collected from infected
plants at the rate of l' to 5%/.(GHmerel' aL, 1974) and such infections
- --".-
-~'-_.
-
-
appeared to provide inoculum for sec~q~ry spread in the field (Gilmer
~
/ '
,~'
et a~., 1974).
In Nigeria, CYMVwas t,(ansmitted in nature mostly by the
beetle Ootheca mutabUis Sahl. (èhrysomel idae) but other vectors were
recorded elsewhere (Gilmer et aL, 1974).
The most promising method to control CYMV is the use of
resistant cultivars and some cowpea varieties developed at liTA exhibited
resistance to CYMV and other cowpea viruses (Singh et a~., 1984;
Williams
1977a, b).
However, these successful results should take into account
the variability (virulence) among CYMV isolates (Gilmer et aL, 1974;
Thottappilly and Rossel, 1984).
11
1.1.5.2
Cucumber mosaic virus: cowpea strain (CuMV)
The cowpea strain of cucumber mosaic virus was regu1ar1y
reported from cowpeas in Nigeria (liTA, 1978;
Robertson, 1966;
Thottappi11y and Rossel, 1984).
Cucumber mosaic virus was transmitted
by sap inoculation and in a non-persistent manner by Aphis craccivoPa
Koch and MYzus persicae Su1z. (liTA, 1978).
A close sero10gica1 re1a-
tionship was found between a Nigerian CuMV isolate and cucumber mosaic
virus (CMV) iso1ates obtained from various crops of temperate regions
(Thottappi11y and Rossel, 1984).
Because of the potential synergistic
association of CuMV with CAbMV or B1CMV, 1eading to considerable yie1d
reductions, this virus constitutes a serious threat for cowpea produc-
tion in many cowpea growing areas (Coll ins et al., 1985; Fischer and
Lockhart, 1976b; Kuhn, 1964; Thottappi11y and Rossel, 1984).
Intensive
screening tests were conducted at liTA to identify sources of resistance
to CuMV in cowpeas.
Severa1 germp1asm accessions were found resistant
to this virus (Thottappi11y and Rossel, 1984).
Cowpea mott1e virus (CMeV)
(1963) first reported the occurrence of cowpea
mott1e virus
wpeas in the western, eastern and southern provinces
of northern Nig
CMeV was further characterized by Shoyinka et al.
(1978).
The v· us was seedborne (but at 10w 1eve1s) in severa1 cowpea
varieties and
t was transmitted by the beet1e Ootheca mutabilis Sahl.
and Medythia quaterna Fairm. (Chrysome1idae) (Allen et al., 1981;
Shoyinka et al., 1978).
CMeV infected Voandzeia subterranea Thouars
and many other species of Leguminosae, Chenopodiaceae and Solanaceae.
It appears to be confined to Nigeria and exc1usive1y in the midd1e be1t
area with a southern guinea savanna climate, where most of the Bambarra
groundnut is grown (Shoyinka et al., 1978;
Thottappi11y and Rossel,
1984).
Susceptible plants reacted with severe ch10rosis, uniform mosaic
or mott1e, local les ions with or without systemic spread, vein necrosis
and symptom}ess infection (Shoyinka et al., 1978).
Multiple infections
inc1uding CMeV, CYMV and SBMV (cowpea strain) were common throughout
cowpea producing areas in Nigeria (Shoyinka et al., 1978;
Shoyinka and
Okusanya, 1975).
12
CMeV is potentially a serious threat to cowpea production
in Nigeria and efforts are being made at liTA to identify cowpea lines
resistant to this virus (Thottappilly and Rossel, 1984).
The results
obtained by screening cowpea accessions for resistance to CMeV isolates
showed sorne breeding lines resistant or tolerant to CMeV (Singh et al.,
1984) .
1.1.5.4
Cowpea golden mosaic virus (CGMV)
A golden mosaic disease was reported from south-east Nigeria
which led to an almost complete loss of the production of cowpea (liTA,
1978).
A virus possessing isometric paired particles (Geminivirus) has
been found associated to the disease (liTA, 1979, 1980).
CGMV was trans-
mitted by the whitefly Bemisia tabaci Gen. and its host range was restricted
to Leguminosae (Anno-Nyako et al., 1983). The vi rus is common in tropical
rainforests in Nigeria (Singh et al., 1984).
Seasonal variation in the
incidence of cowpea golden mosaic virus corresponded closely with fluc-
tuations in population density of its vector (Vetten and Allen, 1983).
ln 1984,during the dry season, CGMV reached epidemic levels in the
northern States of Nigeria.
CGMV constitutes a real threat to cowpea production in Nige-
ria.
Sources of resistance have been identified at liTA (Singh and Allen,
1980;
Thottappilly and Rossel, 1984).
1. 1.5.5
Southern bean mosaic virus: cowpea strain (SBMV-CS)
Southern bean mosaic virus was reported from cowpea in diffe-
rent locations in Nigeria (Shoyinka et al., 1979;
Shoyinka and Okusanya,
1975;
Williams, 1975).
SBMV-CS often occurred with other viruses (CHeV
and CYMV) (Shoyinka et al., 1979).
It induced mosaic and mottle diseases
of economic importance in cowpeas and to a lesser extent in soybean
(Glycine max). Its host range was rather restricted to Leguminosae, with
the exception of Gomphrena globosa L. (Givord, 1981). Isolates of this
virus rarely infected Phaseolus vulgaris L., except for symptomless infec-
tion of the Pinto cultivar, accompanied by localized infections on
inoculated leaves.
13
Ootheaa mutabilis is the important vector of this virus in
Nigeria, but other beetles are also known (Allen et al., 1981;
Shoyinka
et al., 1979).
Seed transmission rates were rather 10w (1-4%) (Shephe rd
and Fulton, 1962).
Yield reductions of cowpea production were not clearly
established in Nigeria.
Givord (1981) in Ivory Coast reported average
yield reductions of 51 to 59%.
The occurrence of mixed infections of CYMV and SBMV at scat-
tered locations in southern Nigeria and the transmission of both vi ruses
by the same vector constitute a serious threat to current efforts to
increase cowpea production in Nigeria.
However, some cowpea breeding
lines exhibited resistance to SBMV and to other cowpea viruses (Singh
et al., 1984).
1.1.5.6
Cowpea mild mottle virus (CMMV)
Cowpea mild mottle virus was first reported from infected
cowpeas in the eastern region of Ghana (Brunt and Kenten, 1973).
Later,
the same virus was observed in cowpeas and soybeans in Nigeria and in
other African countries (Anno-Nyako et al., 1984;
liTA, 1978;
Thottappilly and Rossel, 1984).
Several wild leguminous plants including Calopogonium
muaunoides Desv., Centrosema pubesaens Benth., C. plumieri (Turp. &
Pers.) Benth. and Desmodium saorporius Desv. were natural hosts of CMMV
in Nigeria (Anno-Nyako et al., 1984).
The virus was more common in soy-
bean and in some leguminous weeds than in cowpeas.
Infected plants
remained symptomless or showed mild chlorotic mottle or chlorosis.
The
virus could not be transmitted by aphids (Aphis araaaivora and Myzus
persiaae) (Brunt and Kenten, 1973) but the whitefly Bemisia tabaai Gen.
was found to be an efficient vector (Anno-Nyako et al., 1984).
This
virus does not seem to represent a serious problem for cowpea production
in Nigeria since its occurrence on cowpeas is rare.
1.1.5.7
Tobacco mosaic vi rus: cowpea strain (TMV-CS)
ln 1955, Lister
and Thresh reported a strain of tobacco
mosaic virus (TMV) inducing a systemic disease on cowpea in Nigeria.
14
This observation was confirmed
later (Bawden, 1956;
Chant, 1959, 1960)
but the cowpea strain of TMV was rather rare in cowpeas and it seems like
this virus does not constitute a real prob1em for cowpea production in
Nigeria (Thottappilly and Rossel, 1984).
Many of the viruses listed in Table 1 were transmitted
through seeds from infected plants and such infections appeared to be
the primary source of inoculum for secondary spread in the field by
vectors.
Most of these vi ruses have active vectors (aphids, whiteflies
and beetles) in nature which facilitate their spread from infected plants
to susceptible plants.
For a long time, cowpea yellow, cowpea aphid-borne mosaic
and cowpea mottle virus diseases have been established as the three most
important cowpea diseases in Nigeria (Williams, 1975).
With increasing
virus research on cowpeas in Africa, one can expect to identify other
viruses from this crop in addition to those already reported in Table 1.
1.2
Viruses of the cowpea aphid-borne mosaic virus (CAbMV) complex
1.2.1
Geographical distribution and economic importance of CAbMV
and B1CMV
Cowpea aphid-borne mosaic virus, singly or in mixtures with.
other viruses, causes the most widespread virus diseases of cowpeas in
Nigeria as weIl as elsewhere (Rossel et aL., 1985).
B1CMV and CAbMV are
reported from various different countries where cowpeas are grown (Table
2).
The prevalence of both viruses in cowpeas in various countries in
the world is exp1ained by their seed -- and aphid -- transmission
(Thottappilly and Rossel, 1984).
For CAbMV, crop losses from 13 to 87%
under field conditions and from 29 to 44% after artificial inoculation
in the field, have been reported from Iran (Kaiser and Mossahebi, 1975).
ln greenhouse studies B1CMV reduced the seed yield of Cal ifornia Blackeye
cowpea by 2.5% (Pio-Ribeiro et aL., 1978).
CAbMV was reported to cause
deformation of pods, reduction in size, discoloration of seeds and lower
seed germination.
But there was no correlation between yield reduction
and the percentage of seed transmission (Kaiser and Mossahebi, 1975).
15
TABLE 2.
Geographical distribution of viruses of the cowpea aphid-borne
mosaic virus complex (B1CMV and CAbMV)
Countries
B1CMV
CAbMV
References
AFRI CA
Egypt
+
Anonymous (1981)
Kenya
+
Bock (1973)
Morocco
+
Fischer and Lockhart
( 1976a)
Ni ge ri a
+
Ladipo (1976)
Rossel (1977)
Tanzania
+
Patel and Kuwite (1982)
Uganda
+
Anonymous (1981)
Zambia
+
Anonymous (1981)
AMERI CA
Braz i 1
+
+
Lima et al.
(1981)
Lin et al.
(1981)
South West Pacific Islands
(Samoa and Tonga)
+
Bock and Conti (1974)
United States of America
+
Ande rson (1955a)
Li ma et al.
(1979)
ASIA
China
+
+
Anonymous (1981)
Chang (1983)
Chang and Kuo (1983)
India
+
+
Mali and Kulthe (1980)
Ma 1i et al.
(1981)
+
Sekar and Su10chana (1983)
Indonesia
+
Iwaki et al.
(1975)
1 ran
+
Kaiser and Mossahebi (1975)
Japan
Tsuchizaki et al.
(1970)
The Philippines
+
Beningo and Fava1i-
Hedayat (1977)
AUSTRALIA
+
Behncken and Maleevsky
(1977)
EUROPE
Italy
+
Lovisol0 and Conti (1966)
16
1.2.2
Biological characteristics
1.2.2.1
Sources of inoculum
The primary source of inoculum is constituted by seedlings
infected through the seeds.
The normal practice of sowing seeds randomly
collected, including infected crops, increases the risk of a new out-
break the next year.
As these viruses have many natural aphid vectors
and as isolates of CAbMV or B1CMV can infect other susceptible species of
the Leguminosae family (Anderson, 1955b;
Anonymous, 1981;
Chang, 1983),
inoculum source may be always present in the vicinity of the fields.
1.2.2.2
Transmission through seeds and by vectors
CAbMV and B1CMV are seed-borne in cowpeas.
However, the rate
of seed transmission is usually low (0-3%).
In one case a seed-borne
incidence of 21.5% has been recorded (Tsuchizaki et aZ., 1970).
Seed
transmission of B1CMV or CAbMV is dependent upon the virus strain and
cowpea cultivars and may change from 3.5 to 55% (Gay and Windstead,
1970;
Mal i et al., 1983;
Zettler and Evans, 1972).
From studies
carried out at liTA differences between cowpea accessions were observed
in the rate of seed-infection, ranging from means of 6.3% for TVu 37 to
18.0% for TVx 309-1G (Aboul-Ata et al., 1982).
The severity of symp-
toms and the rate of seed-borne infection were positively correlated.
According to the same studies, there was some indication that seeds
carrying CAbMV were more frequent in early pods than in the late ones.
ln infected dry seeds, CAbMV was located in the cotyledon
and embryo axis.
CAbMV was not recovered from the seed coat of dry
seeds or dry pod tissue whereas CAbMV was recovered from these tissues
at the green mature stage (Ladipo, 1977).
One author found CAbMV par-
ticles in cowpea flowers at three different stages of development and
in young developing pods (Ladipo, 1977).
17
CAbMV and BICMV are readily transmitted, mechanically.
The
following aphids:Aphis craccivora Koch., A. fabae Scop., A. gossypii
Glov. , A. medicaginis Koch., Macrosiphum euphorbiae Thomas and Myzus
persicae Sulz. transmitted these viruses in a non-persistent manner
(Bock, 1973;
Chang, 1983;
Fischer and Lockhart, 1976a;
Vidano and
Conti, 1965).
Screening cowpea germplasm for 'resistance to seed transmis-
sion' could constitute an addi tional means of CAbMV control.
Indeed,
some cowpea lines possessing resistance to seed transmission have been
reported (Aboul-Ata et al., 1982;
Mal i et aZ., 1981, 1983).
1.2.2.3
Host range and symptomatology
BlCMV
Besides cowpea, B1CMV infected other leguminous plants and
sorne species of the following families:
Amaranthaceae, Caesalpinaceae,
Chenopodiaceae, Labiatae and Solanaceae (Table 3) when artificially
inoculated (Chang, 1983;
Mali and Kulthe, 1980).
CAbMV
CAbMV had a relatively wide host range.
This virus infected
experimentally many species and cultivars in the Leguminosae and some mem-
bers of the following families: Amaranthaceae, Caesalpinaceae, Chenopo-
diaceae, Labiatae and Solanaceae (Table 3) (Behncken and Maleevsky, 1977;
Bock
and Conti, 1974).
No species susceptible to both viruses was reported from the
following families:
Apiaceae, Aizoaceae, Comme 1inaceae, Compositae,
Cruciferae, Gramineae, Pedal iaceae and Phytolacaceae (Bock and Conti,
1974;
Chang, 1983;
Chang and Kuo, 1983;
Mali and Kulthe, 1980).
TABLE 3.
Selected susceptible plant species to B1CMV and/or CAbMV
Plant species
B1CMV
CAbMV
References
AMARANTHACEAE
GomphY'ena gtobosa L.
NLL
SU
Behncken and Maleevsky (1977);
Chang
( 1983) ;
Mali and Kulthe (1980)
CHENOPODIACEAE
Chenopodium amaY'anticotoY' Coste & Reyn.
CLL
NLL
Behncken and Maleevsky (1977);
Bock (1973);
Chang (1983);
Mali and Kulthe (1980)
C. quinoa Willd.
CLL
CLL, S
Behncken and Maleevsky (1977);
Bock (1973);
Chang (1983);
Mali and Kulthe (1980)
Spinacia 0 teY'aceae L.
0
CLL,
Behncken and Maleevsky (1977);
Bock
SU
(1973)
LABIATAE
Ocimwn basi t icwn L.
-
NLL
Behncken and Maleevsky (1977);
Bock (1973);
Mali and Kulthe (1980)
LEGUM 1NOSAE
Cajanus cajan Millsp.
0
SU
Bock (1973)
Catopogonium mucunoides Desv.
0
Mo, St
Behncken and Maleevsky (1977)
Canavatia ensiformis (L.) DC
-
0
Mali and Kulthe (1980)
Cassia occidentatis L.
0
Mo
Behncken and Maleevsky (1977)
CentY'osema pubescens Benth.
0
Mo
Behncken and Maleevsky (1977)
-
00
TABLE 3 (continued)
Plant species
B1CMV
CAbMV
References
LEGUMINOSAE (continued)
CrotaZaria brevidens Benth. var.
intermedia (Kotschy) Pohl.
o
S
Bock (1973)
crotaZaria juncea L.
o
S
Bock (1973)
C. pauZina Schrank
o
S
Bock (1973)
C. spectabiZis Roth.
o
S
Bock (1973)
C. usaramoensis Baker
o
Mo
Behncken and Maleevsky (1977)
Desmodium tortuosum (Sw.) D.C.
o
Mo, St
Behncken and Maleevsky (1977)
cyamopsis tetragonoZoba (L.) Taub.
cv.
Pusa Naubahar
o
Mali and Kulthe (1980)
GZycine max (L.) Merr.
Mo
S
Bock (1973);
Chang (1983)
G. max cvs Bragg
Mt
CLL
Behncken and Maleevsky (1977);
Mali and
Kulthe (1980)
Dare
o
CLL
Behncken and Maleevsky (1977)
Lee
o
CLL
Behncken and Maleevsky (1977)
Monetta
CLL, Mo
o
Mali and Kulthe (1980)
lathyrus odoratus L.
o
Nec.
Behncken and Maleevsky (1977)
MacroptiZium atropurpureum (OC) Urb.
o
SU
Behncken and Maleevsky (1977)
M. Zathyroides (L.) Urb.
o
S, STN
Behncken and Maleevsky (1977);
Bock
(1973)
MacrotyZoma uniftorum (Lam.) Verdc.
o
STN
Behncken and Maleevsky (1977)
Medicago scuteZZata
(L.) Mill.
o
ChI.
Behncken and Maleevsky (1977)
\\0
TABLE 3 (continued)
Plant species
B1CMV
CAbMV
Refe rences
LEGUMINOSAE (continued)
Muauna pruriens (L.) D.C.
o
NLL
Bock (1973)
Phaseolus lunatus L.
Sev. Mo
Mi 1d
Behncken and Maleevsky (1977);
Mali and
ChI .•
Kulthe (1980)
Mt.
Phaseolus vulgaris L.
o
NLL. S
Bock (1973);
Chang (1983)
Phaseolus vulgaris L. cvs
Biela Kockova
CLL. Mo
0
Mali and Kulthe (1980)
Per 1i cka
CLL
0
Mali and Kulthe (1980)
Black Turtle
Mo. NLL
Behncken and Maleevsky (1977);
Chang
(1983);
Fischer and Lockhart (1976a)
Bount i fu 1
Mo
Mil d
Behncken and Maleevsky (1977);
Chang
Mt
(1983)
Pinto
Mt
CLL. Mt
Behncken and Maleevsky (1977);
Mali and
Kulthe (1980)
Prince
Mo. NLL
0
Mali and Kulthe (1980)
Pisum sativum L.
S
Bock (1973);
Chang (1983);
Mali and
Kulthe (1980)
P. sativum cvs Dark Skin
o
SLI
Behncken and Maleevsky (1977)
Perfection
o
SLI
Behncken and Maleevsky (1977)
..
Vanco Choice
o
SLI
Behncken and Maleevsky (1977)
Sesbania speaiosa Taub. ex. Engl.
o
Mo
Behncken and Maleevsky (1917)
N
Triforium inaarnatum L.
o
S. SLI
Behncken and Maleevsky (1977);
Bock (1973)
o
T. subterraneum L.
o
S
Bock (1973)
Viaia faba L.
NLL
Behncken and Maleevsky (1977);
Chang (1983)
TABLE 3 (continued)
Plant species
BICMV
CAbMV
References
LEGUMINOSAE (continued)
V. faba
L.
cv. Slovak Local
o
Mali and Kulthe (1980)
Vigna mungo (L.) Hepper
Mo
Behncken and Maleevsky (1977);
Chang
( 1983)
V. mungo cv. Sindkheda-I-I
o
Mali and Kulthe (1980)
V. radiata (L.) Wi Icz.
o
SI
Bock (1973)
V. radiata cvs Jalgaon-781
Mt, NLL
o
Mali and Kulthe (1980)
VC.
Tainan No. 1
Mt
o
Chang (1983)
V. unguicuZata (L.) Walp. ssp.
sesquipedaZis
o
Mo
Behncken and Maleevsky (1977)
V. unguicuZata (L.) Walp. ssp.
sesquipedaZis cv. Kao Hsiung
green pod
Vb, Mo
Mo
Chang (1983);
Chang and Kuo (1983)
V. unguiauZata (L.) Walp. ssp.
Mo
S, Mo
Behncken and Maleevsky (1977);
Bock (1973);
unguicuZata
Mali and Kulthe (1980)
·v. unguicuZata (L.) Walp. ssp.
unguiauZata cvs Blackeye
Mt
o
Mali and Kulthe (1980)
California Blackeye
Mt
Mt
Chang (1983);
Chang and Kuo (1983)
C-152
Mt
o
Mali and Kulthe (1980)
CG-28
Mt
o
Mali and Kulthe (1980)
CM-II
Mt
o
Mali and Kulthe (1980)
CoPusa-4
Mo, Vb
o
Mali and Kulthe (1980)
N
Early Ramshorn
Mo, Vb
o
Mali and Kulthe (1980)
TABLE 3 (continued)
Plant species
B1CMV
CAbMV
References
SOLANACEAE
Nicotiana benthamiana Domin.
Mt
Mo
Chang (1983);
Chang and Kuo (1983);
Thottappilly and Rossel (1984)
N. cZeveZandii Gray
Mi Id
Benhcken and Maleevsky (1977);
Bock (1973);
ChI.
Mali and Kulthe (1980)
Mt
N. T'Ustica L.
SLI
Behncken and Maleevsky (1977);
Chang
( 1983)
Petunia hybrida Vilm.
SLI
Behncken and Maleevsky (1977);
Chang
(1983)
CLL:
Chlorotic local les ions
Ch 1. :
Chlorosis of inoculated leaves
Mo:
Mosaic
SeVe Mo:
Seve re Mesa i c
Mt:
Mottling
Nec. :
Necrosis of inoculated leaves
~LL:
Necrotic local les ions
S:
Systemic infection with symptoms (no details)
SI:
Symptomless systemic infection or systemic infection without symptoms
SLI :
Symptomless local infection
STN:
Stunting
TN:
Tip necrosis (no details)
Vc:
Vein clearing
VY:
Ve in ye 11 owi ng
0:
Not tested
Not susceptible
N
N
23
Most studies on CAbMV and/or B1CMV have shown variation in
host reactions between different isolates (Bock, 1973;
Ladipo and Allen,
1979a;
Taiwo et al., 1982).
There were some obvious disparities in the
host range studies with CAbMV.
An Italian isolate of cowpea aphid-borne
mosaic virus was found to cause symptoms in Gomphrena globosa L. and
Pisum sativum L. (Lovisol0 and Conti, 1966).
But a Moroccan isolate of
the same virus did not infect these two plants (Fischer and Lockhart,
1976a).
Lovisol0 and Conti (1966) could infect Chenopodium foetidum
Schrad., Cuaumis sativus L., Cuaurbita pepo L., Petunia hybrida Vilm.,
Phaseolus lunatus L., Physalis alkekengi L., Physalis floridana Rydb.
and Niaotiana tabaaum L. cv. White Burley with their isolate of CAbMV
from Italy.
These authors,as weIl as Bock and Conti (1974), described the
local reaction of Ocimum basiliaum L. as of diagnostic value.
However,
repeated attempts to obtain this kind of reaction on this species failed
with a Moroccan isolate of CAbMV (Fischer and Lockhart, 1976a).
Several cultivars of French bean (Phaseolus vulgaris L.)
were susceptible to CAbMV infection but here also there were some dis-
crepancies (Behncken and Maleevsky, 1977;
Bock, 1973;
Lovisol0 and
Conti, 1966).
Most bean cultivars were not susceptible to systemic
infection with an Australian isolate of CAbMV, although many developed
chlorotic lesians on inoculated primary leaves.
Some other cultivars
were susceptible and developed severe systemic symptoms.
These were
chlorotic or necrotic local lesions, followed by spreading vein and
petiole necrosis and epinasty with wilting distortion and green mottle
of the new foliage (Behncken and Maleevsky, 1977).
The host range and
symptomatology of the 'Queensland' isolate of CAbMV was, in general,
quite similar to those reported for other isolates (Behncken and Maleevsky,
1977;
Bock, 1973;
LovIsol0 and Conti, 1966).
However it did not cause
systemic infection in Glyaine max (L.) Merr., Pisum sativum L. and
Petunia hybrida Vilm., three test plants described as diagnostic
species by Bock and Conti (1974).
Ladipo (1976) and Kaiser and Mossahebi (1975) reported that
Niaotiana glutinosa L. became infected by their isolates of CAbMV but
this result could not be confirmed by other authors (Fischer and
Lockhart, 1976a;
Mali and Kulthe, 1980).
The dangers of relying only
24
on host range and symptomatology for identification of CAbMV and/or B1CMV
are illustrated by the apparent inability of an isolate M (described by
Bock, 1973) to infect many
species susceptible to other strains (Tl, T2
and VB1) (Bock, 1973).
After careful analysis of the results obtained by different
workers, there is some evidence that no single test plant reaction could
be described as strictly diagnostic for aIl strains or isolates of CAbMV
and/or B1CMV.
Some of the discrepancies are undoubtedly due to host
plant variation, variability of virus strains or isolates and possibly
mixed infections (CAbMV and CuMV).
EXTERNAL SYMPTOMS
When B1CMV or CAbMV is inoculated to cowpeas, four types of
reactions may be observed.
1.
Local reactions which may consist of necrotic local
lesions, chlorotic local lesions, necrotic spots, spots or necrosis on
primary leaves (Behncken and Maleevsky, 1977;
Lovisolo and Conti, 1966;
Mali and Kulthe, 1980).
2.
Systemic reactions, consisting of mosalc, mottle, vein-
banding (with or without reduction in leaf size), vein clearing, and
veinaI chlorosis (Bock and Conti, 1974;
Mali and Kulthe, 1980).
3.
A combinat ion of the two previous types of symptoms
(Bock, 1973;
Chang, 1983).
4.
Symptomless local and systemic reactions (Bock, 1973;
Mali and Kulthe, 1980).
2S
5ymptoms, induced by CAbMV or BICMV, current1y reported on
cowpeas in the 1iterature are summarized be10w.
With an Indian iso1ate
of B1CMV, the symptoms on primary 1eaves of CM-11 cowpea, were mi1d
mosaic fo110wed by irregu1ar mosaic or ye110w mott1e, puckering, slight
distortion, and arching of trifo1iate 1eaves.
51 ight stunting of the
plants was a1so observed (Mali and Ku1the, 1980).
B1CMV produced both
local and systemic symptoms on B1ackeye cowpea.
Local symptoms inc1uded
rather large reddish and often ring-1ike 1esions which spreaded a10ng the
veins, forming a reddish net pattern.
5ystemic symptoms consisted of
severe mott1ing distortion and some reddish vein necrosis.
5ystemic
symptoms on other cultivars inc1uded mott1e in which green bands a10ng
the veins common1y a1ternated with chlorotic or with interveina1 areas
together with some stunting and distortion (Anderson, 1955a;
Anonymous,
1981;
Mal i and Ku1 the, 1980).
Cowpea plants infected with CAbMV showed a prominent mosaic
on most 1eaves, together with 1eaf distortion and reduction in 1eaf
size.
The proportion of ye110w 1eaf tissue and the severity of distor-
tion varied with cultivars and virus strain (Behncken and Ma1eevsky,
1977).
Diseased cowpea plants infected with CAbMV showed variable
amounts of dark green veinbanding or interveina1 ch10rosis, 1eaf dis-
tortion, b1istering and stunting (Bock and Conti, 1974).
INCLUSION BOVIES
Iné1usion bodies, either in the cytop1asm or in the nucleus,
are of special interest because they often consist of special structures
characteristic of infection by certain vi ruses (ex. pinwhee1s of poty-
vi ruses) (Christie and Edwardson, 1977).
These structures can readi1y
be detected with the light microscope.
Light microscopic observations
of epiderma1 1eaf strip preparations from cowpea plants systemica11y
infected by B1CMV (Lima et aZ., 1979) revea1ed tubu1ar cytop1asmic
inclusions and cy1indrica1 inclusions simi1ar to those reported for
B1CMV in other hosts (Christie and Edwardson, 1977).
In sections of
cowpea leaves infected by CAbMV cy1indrica1 inclusion bodies characte-
ristic of the potyvirus group are frequent1y seen in the cytop1asm by
e1ectron microscopy (Behncken and Maleevsky, 1977;
Lima et aZ., 1979).
These intrace11u1ar inclusions are sometimes useful for diagnosis of
26
vi ruses belonging to the potyvirus group.
Even if they are diagnostic
of the potyvirus group, these inclusions cannot be used to define any
grouping among strains or isolates of B1CMV or CAbMV.
1.2.3
Strains and/or isolates described in the literature
Four major strains (European type, African neotype, African
mild and African veinbanding strains) of CAbMV have been distinguished
according to the systemic symptoms induced on cowpea Mak 1 (Bock, 1973).
ln addition to these four strains, several isolates of
CAbMV and/or BICMV have been described throughout the world which were
mostly different in host range characteristics.
For example, the host
range and symtomatology of an Australian isolate (Behncken and Maleevsky,
1977), is
quite
similar
to
those reported for other isolates
(Bock, 1973;
lovisolo and Conti, 1966).
But, it did not cause infec-
tion in Glycine max, Pisum sativum and Petunia hybrida.
These hosts
have been described as diagnostic species for CAbMV by Bock and Conti
(1974).
Most studies on CAbMV have shown variation in host reactions
between different isolates (Behncken and Maleevsky, 1977).
The same
observations were also reported with BICMV isolates (Taiwo et al.,
1982).
1.2.4
Separation of vi ruses from mixed infections (combinations
of CAbMV, BICMV and CuMV or other cowpea vi ruses)
ln nature, frequent mixtures of sorne of these viruses occur·
in cowpeas.
Unless mixed infections can be separated into single enti-
ties, it is difficult to conduct comparative studies (Kuhn, 1964).
Since different vi ruses may cause diseases of cowpeas in Nigeria, dual
or multiple infections in various combinations are possible (Thottappilly
and Rosse l, 1984).
Sorne reports of synergistic interactions resulting in high
losses include:
1.
a disease complex of cowpeas caused by strains of CuMV
and CAbMV in Morocco (Fischer and Lockhart, 1976a, b);
27
2.
a stunt disease caused by the combination of B1CMV with
CuMV in the U.S.A. (Kuhn, 1964) and in China (Chang, 1983).
Various types of strains may be recovered from infected
plants.
In order to obtain pure virus isolates from mixtures, it is
necessary to develop separation techniques.
Some are described below
for separation of cowpea viruses from mixtures.
1.2.4.1
Inoculation of differential test plants
Separation may be accomplished simply by inoculating a number
of different test plants followed by back inoculation to the original
species from which the virus was isolated.
This technique is especially
useful when a test plant developed local lesions with one of the viruses
in the mixtures and others did not systemically infect this same test
plant.
Fischer and Lockhart (1976a) used Glyaine max to separate CAbMV
from CuMV when they occurred as mixed infections.
Chang (1983) and Pio-
Ribeiro et al. (1978) recommended Cassia obtusifolia L. or Cassia topa L.
for the same purpose (to separate B1CMV and CuMV) because obvious local
and systemic symptoms developed on C. obtufisolia as a result of infec-
tion with B1CMV.
Infection of Glyaine rn::lX with B1CMV was erratic and
was therefore less recommended (Fischer and Lockhart, 1976a).
Single les ions can be transferred and this can be repeated
several times for purposes of biological purification of viruses
(Boxhall and MacNeill, 1974).
Since each virus may exist as a family of
more or less closely related 'strains' or forms, a particular isolate
derived from a single local les ion might be useful for specific studies.
However, recent results shed suspicion on the reliability of local
lesions passage as a means of getting biologically 'pure virus' (Garcia-
Arenal et al., 1984).
First,contaminants could be passaged along with
the major virus (normally they would not be detected, but they might be
expressed under the appropriate environmental
conditions) (Bawden,
1956;
MacNeill and Boxhall, 1974).
Second'y, high rates of infidelity
noticed during the transcription
process
generate~ heterogeneous viral
RNA s even in an appa ren t 1Y homogeneous 1oca 1 1es ion
popu 1a t ion (Ga rc i a-
Arenal et al., 1984).
28
Reactions of cowpea viruses in certain hosts can be useful
for tentative identification.
However. caution must be exercised because
different strains may give different reactions whereas other cowpea
vi ruses may produce similar reactions.
1.2.4.2
DifferentiaI transmission br vectors
One virus in a mixture may be transmitted by a specifie vec-
tor (aphids) and this virus can be then readily separated.
For example.
CAbMV and/or B1CMV could be easily separated frorn a mixture containing
CYMV which is transmitted only by beetles.
1.2.4.3
SeriaI transmission br a vector
A vector carrying more than one virus may transmit separa-
tely one of the viruses to some plants after seriaI feedings.
By this
means, Chang (1983) succeeded in separating B1CMV from CuMV in asparagus
bean (Vigna unguiculata (L.) Walp. ssp. sesquipedalis).
1.2.4.4
Seed transmission
A seed transmission test could be used to separate B1CMV and
CuMV (Pio-Ribeiro et al •• 1978).
ln addition to separation of B1CMV and/or CAbMV from un-
related viruses, it is necessary to separate B1CMV from CAbMV if they
occur in a mixture.
Cowpea lines, resistant to each virus, have been
identified (Ladipo and Allen, 1979a;
Patel et al., 1982;
Taiwo et al .•
1982).
These resistant lines can be used in separating CAbMV and B1CMV
from mixed infections.
According to Taiwo et al.
(1982), these resis-
tant cowpea lines would be a valuable tool for diagnostic purposes to
identify B1CMV and CAbMV isolates.
1.2.5
Identification of sources of resistance
The identification of cowpea cultivars resistant to one or
both viruses constitutes an important step towards developing an ade-
quate control measure.
Studies by several workers on sources of resis-
29
tance to B1CMV and/or CAbMV revea1ed the existence of many resistant
cowpea lines (Ladipo and Allen, 1979a;
Lima et aL, 1979;
Pate1 et al.,
1982;
Taiwo et al., 1981, 1982).
Resu1ts showed
a
range
of
reactions of the cowpea 1ines vis-à-vis the different iso1ates of B1CMV
and/or CAbMV.
Immunity, to1erance, moderate resistance and susceptibi-
lity were the reactions recorded (Ladipo and Allen, 1979a;
Pate1 et al.,
1982) .
Two aspects of the eva1uation of cowpea 1ines for resistance
to B1CMV and/or CAbMV will be considered here.
1.2.5.1
The eva1uation of germp1asm accessions
The eva1uation of germp1asm accessions for resistance to
cowpea virus diseases may provide va1uab1e information for breeders in
their effort to identify good virus resistance.
For this purpose, the
TVu 1ine 128 (TVu = Tropical Vigna unguiculata) was found as a good
virus resistance donor (Rossel and Huttinga, 1983).
Other lines (TVu
612, 2480 and 2657) expressed immunity reactions to at 1east three out
of four iso1ates of CAbMV (iso1ates from Kenya, Nigeria, Tanzania and
the United States of America) (Ladipo and Allen, 1979a;
Pate1 et al.,
1982;
Taiwo et al., 1982).
Various TVu 1ines, tested by Taiwo (1978), (TVu 222, 612,
1948, 1258-1, 1330, 1453, 1630,2480, 2657) were found immune to the
Nigerian iso1ate of CAbMV.
But some of these immune 1ines, 1ike TVu
1948 or TVu 1630, were susceptible to the Kenyan iso1ate of CAbMV.
This fact revea1s again the discrepancies in these resistance studies
and suggests variation among the iso1ates tested (Ladipo and Allen,
1979a;
Taiwo et al., 1982).
1.2.5.2
The eva1uation of advanced breeding 1ines
ln addition to the screening of the germp1asm accessions for
resistance to B1CMV and/or CAbMV, advanced breeding. 1ines were screened
for resistance to the same viruses.
The resu1ts indicated that the
fo11owing breeding 1ines: IT82E-16, IT82D-807, IT82D-808 and IT82D-950
30
possess a moderate to high levels of resistance to CAbMV and other cow-
pea virus diseases (Singh et al., 1983).
Several reports were publ ished
on the inheritance of resistance to B1CMV and/or CAbMV indicating that
resistance to B1CMV is controlled by a single recessive gene (Taiwo et
al., 1981).
According to the same authors, the cowpea lines TVu 2740,
TVu 3273, TVu 2657 and TVu 2845 seem to possess this same gene.
For
CAbMV, the resistance is controlled by two pairs of recessive genes
(S i ngh et al., 1984).
1.2.6
Physico-chemical properties
Resistance studies on viral diseases require knowledge of
the vi ruses concerned and of their strains (isolates).
This implies
studies of the biological as weIl as physico-chemical properties.
For
study of some of these characteristics, purified virus preparations
are req ui red.
1. 2 . 6. 1
Pur i f i ca t ion
The following hosts have been commonly used for purification
of B1CMV and/or CAbMV:
Vigna unguieulata and Nicotiana benthamiana
Domin (Bock, 1973;
Lima et al., 1979;
Taiwo et al., 1982).
Routinely,
both vi ruses are propagated in Vigna unguieulata (Behncken and Maleevsky,
1977;
Lima et al., 1979;
Taiwo et al., 1982).
The extraction process
is made with a sodium citrate buffer (0.5M, pH 8.1) (Bock, 1973) or
phosphate (0.5M, pH 7.5) (Lima et al., 1979;
Taiwo et al., 1982).
Stabilizing additives, such as 2-mercaptoethanol, ethylene diamine
tetraacetic acid (di-sodium salt) (EDTA) and sodium sulfite (Na2S03) are
commonly used.
Clarification is usually performed for both viruses with
either n-butanol (8%, v/v) or a combination of chloroform and carbon
tetrachloride (1: l, v/v) (Lima et al., 1979;
Taiwo et al., 1982).
Virus particles are then concentrated by differential cen-
trifugation.
Further purification requires the use of sucrose density
gradient (Bock, 1973) or equilibrium density gradient centrifugation in
cesium chloride (Cscl) or cesium sulfate (CS 2S0 3) (Lima et aL, 1979;
Taiwo et al., 1982).
31
1.2.6.2
Morphology and particle sizes
Electron microscope observations of partially purified or
leaf dip preparations from cowpea leaves, infected by B1CMV or CAbMV
isolates, showed flexuous particles (Behncken and Maleevsky, 1977;
Bock
and Conti, 1974;
Ladipo, 1976;
Lima et al., 1979;
Taiwo et al.,
1982).
For B1CMV, a modal length of 753 nm was reported (Lima et al. ,
1979).
The mean width of the virus partic1es was 13 nm for B1CMV and
11 nm for CAbMV (Boswell and Gibbs, 1983).
Taiwo et al.
(1982) measured numerous particles of both
viruses and found that (for each isolate) 60 to 81% of the particles had
1ength ranging from 720 to 760 nm with a mean of 743 nm.
The same
authors studied the stability in sap of different isolates of B1CMV
and CAbMV.
The results indicated a thermal inactivation point (TIP)
between 57 and 600C, 10ngevity in vitro (LIV) at 220 C between 24-48 hours
and a dilution end point (DEP) between 10- 4 and 10- 5
(Taiwo et al.,
1982).
There was no marked difference
between these characteristics
with both viruses.
Virus partic1es of B1CMV or CAbMV sediment as one component
and the values of their sedimentation coefficient were 150S for CAbMV
and 157 to 159S for B1CMV (Bock and Conti, 1974;
Lima et al., 1979).
The genome for both vi ruses is a single-stranded 1inear RNA with a sedi-
mentation coefficient of 40S (Taiwo et al., 1982).
Only one protein
species exists in the virus particles of B1CMV and CAbMV (Taiwo et al.,
1982).
The molecular weights (MW) of the viral coat protein of B1CMV
and CAbMV were estimated at 34,000 d and 30,000 to 35,000 d respectively
(Lima et al., 1979;
Taiwo et aL, 1982).
The oost important physico-
chemical properties of both viruses are summarized in Table 4.
1.2.7
Antigenic properties of the virus
ln general, potyviruses react wel1 in a number of sero10gical
tests.
Sorne of these, current1y used for the characterization of B1CMV
and CAbMV isolates, are presented below.
32
TABLE 4.
Comparison of physico-chemical properties of BICMV and CAbMV
Vi rus
BICMV
CAbMV
References
particles
TIP
57-600C
57-600 C
Bock and Conti (1974)
LIV
1-2 days
1-2 days
Bock and Conti (1974)
Lima et al. (1979)
Taiwo et al. (1982)
DEP
10-1+ to 10- 5
10- 3 to 10- 5
Taiwo et al. (1982)
MW
34,000 d
30,000 to
Taiwo et al. (1982)
35,000 d
Lima et al. (1979)
Sv
157-1595
1505
Taiwo et al. (1982)
Bock and Conti (1974)
RNA (%)
5-6
5-6
Boswe11 and Gibbs (1983)
RNA (5)
405
405
Taiwo et al. (1982)
Particle
sizes
13 x 753 nm
11 x 750 nm
Boswe11 and Gibbs (1983)
NOTE:
For detai Is, see section 1.2.6.2
1.2.7.1
Microprecipitin test
This test is often used by different workers for the deter-
mination of antisera titers and sero10gica1 inter-re1ationships among
CAbMV and B1CMV isolates and other potyviruses infecting cowpeas
(Behncken and Ma1eevsky, 1977;
Bock, 1973;
Ladipo, 1976;
Mali and
Ku1 the, 1980).
Bock (1973) in his studies on the serological re1ationships
of the East African CAbMV iso1ates made the fo110wing observations.
Three strains: neo-type (Tl), veinbanding (VBI) and mi1d (M) cou1d be
differentiated.
Within the African strains, the neo-type and mi1d
strains are sero10gical1y identica1.
Fina11y, the veinbanding strain,
a1though re1ated to the neo-type and the mild strain, is sero10gica11y
distinct.
33
1.2.7.2
SDS - immunodiffusion tests
Double diffusion tests in SDS gels have been used success-
fully for diagnosis and comparative studies (e.g. determination of
relationships) of CAbMV and B1CMV isolates (Lima et al., 1979;
Taiwo
and Gonsalves, 1982).
Using the intragel cross-absorption technique,
lima et al.
(1979) showed that BCMV and e i ther CAbMV or BI CMV were
serologically distinct.
Recent studies comparing different isolates of B1CMV and
CAbMV revealed that both viruses, although serologically related, are
not identical (Taiwo and Gonsalves, 1982).
AlI the se results confirmed previous results showing that
B1CMV is very closely related to BCMV (Bock, 1973;
Bock and Conti,
1974).
However, a closer serological relationship exists between B1CMV
and BCMV than between B1CMV and CAbMV (Lima et al., 1979).
Discrepancies
were
noticed
in
some
of
the
results
obtained by different workers.
lima et al.
(1979) found stronger hete-
rologous reactions between their antisera to B1CMV and CAbMV.
Their
antiserum to B1CMV cross-reacted with several other potyviruses among
which legume viruses such as soybean mosaic virus (SHV) , bean yellow
mosaic virus (BYMV) and BCMV.
On the other hand, the findings of Taiwo
and Gonsalves (1982) and Bock (1973) disagree with the results of Lima
et al.
(1979) on that particular point.
1.2.7.3
Enzyme-linked immunosorbent assay (ELISA)
The enzyme immuno-assay technique developed by Clark and
Adams (1977) is very sensitive and increasingly used to detect plant
vi ruses especially those at low con-centrations such as B1CMV or CAbMV
in cowpeas.
This method was used by Taiwo and Gonsalves (1982) to cha-
racterize different isolates of B1CMV and CAbMV from different geogra-
phical areas.
They improved the sensitivity of the test by adding O. lM
34
EDTA to the standard ELISA buffer and using O. lM potassium phosphate
(pH 7.5) as the extraction or dilution buffer.
They performed ELISA
tests using antisera prepared to four isolates of B1CMV and two isolates
of CAbMV.
Different isolates could be classified in two groups:
B1CMV
and CAbMV.
No cross reactions were detected between the two groups.
1.2.7.4
Immune electron microscopy
This method, reviewed by Milne and Luisoni (1977), was used
by Taiwo and Gonsalves (1982) in their comparative studies of B1CMV
isolates.
Their results showed that homologous antisera decorated
heavily virus particles of identical isolates or quite similar isolates.
Only moderate decoration was observed with heterologous antisera and no
decoration was noticed with unrelated virus like pa paya ringspot virus,
using both antisera (Taiwo and Gonsalves, 1982).
Finally, the results obtained with this technique suggested
that it may be more sensitive in detecting low levels of cross reacting
antibodies than SDS-immunodiffusion tests and ELISA (Taiwo and Gonsalves,
1982).
CHAPTER 2
MATERIALS AND METHODS
2. 1
General approach
The main objective of the present study was to collect and
identify different types of CAbMV complex and to differentiate them on
the basis of biological, serological and physico-chemical properties.
The main lines along which this study was conducted are the following:
1.
A survey of different cowpea production areas in
Nigeria for the prevalence of CAbMV and/or B1CMV.
2.
A set up of a collection of different isolates of these
viruses with prior separation fram other vi ruses occurring in mixed
infections on cowpeas.
3.
Partial characterization of these isolates by:
a.
their symptomatology on cowpeas and various test
plant species (artificial host range);
b.
their serological properties and relationships
(SDS- i mmunod i ffus ion in aga r ge 1,5) ;
36
c.
the comparison of the different types of these vi ruses
on liTAIs improved cowpea cultivars and a selection
of other cowpea germplasm accessions.
The various steps of the present studies are summarized in
Figure 2.
2.2
Test plants
AlI plants were grown and maintained at ambient temperature
ranging from 23-360 e in an insect-proof screenhouse designed by Rossel
and Fergu~on (1979).
Seeds of the leguminous plants were sown in unsterilized top
soil in plastic pots (13 cm diameter by 13 cm high).
AlI other plants
were sown in trays containing steril ized soil unless otherwise stated
and maintained in growth chambers (at 250 e (day) and 200 e (night)) until
transplantation in trays or in plastic pots.
Humidity was 81% (day) and
87% (night).
Plants were sprayed weekly with an insecticide dimethoate
at the concentration of 0.15% (Rogor solution with 40% active ingredient).
2.3
Survey
Both farmers' fields and experimental plots in Nigerials
major cowpea producing areas were surveyed for plants showing symptoms
of virus (mosaic, veinbanding).
One trifoliate leaf was collected from
each presumably virus infected plant and stored in tubes with one drop
of water or in plastic bags kept in a cool box for one or two days.
Plants without symptoms (possible latent infections) were also collect-
ed.
Some samples were also collected from wlld cowpea (Vigna unguiauZata
(L.) Walp. ssp. dekindtiana) and from other leguminous crops:
African
yam bean (BphenostyZis stenoaarpa) (Hochst ex. A. Rich) Harms and Bam-
barra groundnut (Voa~eia subterranea Thouars or Vigna subterranea
(Thouars) Verdc.).
APHID TRANSMISSION
SEROLOGY
ELECTRON MICROSCOPY
CMeV, CYMV
' .
< ' j
VIGNA UNGU~CU~TA 1+1 GLYCINE MAX 1+1 CASSIA TORA 1 +1 OTHER TEST PLANTS
SYMPTOMATOLOGY
PURIFICATION 1
1 COMPARATIVE SEROLOGY
-
(SDS-IMMUNODIFFUSIO~
(HOST RANGE)
DIFFERENTIAL REACTIONS ON
COWPEA ACCESSIONS AND
BREEDING
L1NES
APHID TRANSMISSION 1_
1
SEED TRANSM 1SS ION
1
_ ,
\\N
....,
Figure 2:
Outline of present studies
38
2.4
Identification of isolates
To identify and to separate the isolates from other cowpea
viruses, the following methods were used:
1.
electron microscope examination (leafdip preparations);
2.
symptomatology on selected test plants;
3.
serology (immunodiffusion tests and enzyme-linked
immunosorbent assay).
2.4.1
Electron microscope examination
To detennine the morphology of virus particles, leaf crush
preparations were made of foliar tissue.
Small pieces of leaf tissue
were crushed in a few drops of 0.2% sodium sulfite (w/v) in distilled
water.
Sodium sulfite is used as a reducing agent.
Droplets of the
greenish extract were put on parafilm.
Copper grids (300 mesh) , with a
carbon film, were floated on droplets for about one minute after which
the grids were washed several times with distilled water and drained by
touching the edge of each grid to a filter paper.
They were stained
with 2% PTA (phosphotungstic acid, pH 6.8) for about two minutes and
drained by touching the edge of each grid to a filter paper.
The same
procedure was followed with purified virus preparations.
Specimens
were examined with a Philips (EH 201) transmission electron microscope.
2.4.2
Symptomatology on selected test plants
Leaf samples from diseased cowpea plants, collected during
field surveys, were homogenized in a steri lized mortar and pestle in
ice cold buffer (O.OIH potassium phosphate, pH 7.5).
The extract was
rubbed onto one week old cowpea seedlings (cv. Ife Brown) dusted with
600 mesh silicon dioxide (carborundum).
Healthy seedlings inoculated
only with buffer served as controls. Inoculated leaves were immediately
washed with tap water.
The inoculated plants were kept in an insect-
proof screenhouse for observations.
39
When an isolate caused systemic infection on Vigna unguiau-
tata cv. Ife Brown, detailed host range studies and serological tests
were conducted toidentify the virus(es) involved.
For preliminary host
range studies, the following test plants were used:
Arachis hypogaea
L., Chenopodiwn amaranticolor, Glycine max, Gorrrphrena globosa, Nicotiana
benthamiana, N. glutinosa and Ocimwn basilicum.
Usually five individual
plants of each test plant species
were used.
Sorne cowpea lines reslstant to each virus, were also tested
in order to identify the isolates as CAbHV or as B1CHV.
This was
suggested by Taiwo et al.
(1982).
2.4.3
Preliminary serological studies
Serological tests were performed in 0.8% agar plates made up
in disti 11ed water according to the double diffusion technique of
Ouchterlony (1962).
The antisera against the following isometric vi ruses
-- CHeV, CYHV, CuHV and SBHV -- were used.
Enzyme-linked immunosorbent
assay (ELISA) was also used for the identification of the isolates accord-
ing to the procedure described elsewhere (Clark and Adams, 1977;
Taiwo and Gonsalves, 1982;
Thottappilly,1984).
In this case, the anti-
sera from the laboratory (Virology Unit) at lITA against the following
viruses -- CHHV, CAbHV, CYHV and CuHV -- were used.
2.5
Isolates and their maintenance
Eight of B1CHV and/or CAbHV isolates characterized during
present studies are listed in Table 5.
They were propagated in cowpea
plants (Ife Brown), by successive mechanical transfers.
To maintain
the purity of the isolates, aphid transmission was performed at regular
intervals to distinguish from beetle - transmitted viruses, CYHV, CHeV
and SBHV.
To check for contaminating CuHV, isolates were routinely
inoculated to Niaotiana glutinosa.
The isolate 'ONNE' is from the 10cality ONNE near Port-
Harcourt (South Eastern Nigeria in the Rivers State)~
The isolate
40
TABLE 5.
Or igin of the virus iso1ates
Iso1ates
Original host
Loca1ities (state)
ONNE
Vigna unguicu Zata
ONNE (Rivers)
ZARIA
Vigna unguicuZata
ZARIA (Kaduna)
(TVx
3433-02F)
WI LD COWPEA
Vigna unguicuZata
IBADAN (Oyo)
(or WC)
(Wi Id cowpea)
1T 11
Vigna unguicuZata
IBADAN (Oyo)
( 1T 82E-10)
IT 15
Vigna unguicuZata
IBADAN (Oyo)
( 1T 83D-285)
1T 16
Vigna unguicuZata
IBADAN (Oyo)
(IT 83D-262)
BT-4
Vigna suhterranea
IBADAN (Oyo)
AT-5
SphenostyZis stenocarpa
1BADAN (Oyo)
'Zaria' was co11ected from infected cowpea, TVx 3433-02F at Zaria
(Northern Nigeria, Kaduna State) in experimenta1 plots.
The fo11owing
cowpea iso1ates
-- 'IT 11', 'Wild cowpea or WCI, liT 15', liT 16',and
Bambarra groundnut iso1ate, 'BT-4 1 and African yam bean iso1ate,
lAT-5 1 -- are from the liTAis main site at Ibadan (Oyo State).
The isolate liT 11' was origina11y a mixture of CAbMV and
CuMV and was designated liT l1M I .
The filamentous CAbMV (liT 111) was
separated from the isometric virus contaminant (CuMV) by passing through
Cassia tom.
2.6
Artificia1 host range and symptomato1ogy
Leaves from systemica11y infected Vigna unguicuZata cv. Ife
Brown were ground in a co1d steri1ized mortar with O.OlM phosphate buffer
41
(PB)
(pH 7.5) and inoculation was done by standard procedures (see
2.4.2) .
Five seedlings were inoculated with the virus isolates.
The
inoculated plants were kept in an insect-proof screenhouse and symptoms
scored on the different test plants.
2.7
DifferentiaI reactions on various cowpea germplasm accessions and
breeding lines
Fort y promlslng breeding 1ines were tested for their reactions
to the following isolates 'ONNE' t 'IT Ill, 'IT 16 1 and 'Zaria'.
Germplasm
accessions showing resistance to four of these isolates
were re-
evaluated using at this time the eight
CAbMV
and/or B1CMV isolates.
After inoculation, the plants were sprayed weekly with dimethoate.
The
reactions of the plants were scored between seven and thirty days after
inoculation for resistance or susceptibility by assessing the following:
1.
number of infected plants
and
t
2.
intensity of symptoms using a grade scale (1 to 5).
The score 1 denotes no visible symptoms and 5 for very severe symptoms.
Observations were made on inoculated primary leaves (local reactions)
as weIl as on trifoliate leaves (systemic reaction).
Back inoculations
were do ne for the 1ines showing no visible symptoms (score 1) using the
susceptible cultivar Ife Brown.
2.8
Transmission studies
2.8.1
Aphid transmission"
Aphids (Aphis craccivora Koch) were reared on healthy cowpea
(Ife Brown).
They were starved for four to six hours and then transferred
to Petri dishes containing leaves of cowpeas infected by the different
isolates using a moistened hair brush.
The aphids were permitted an
acquisition period of approximately one minute (observed with an eye
lens) and then
ten aphids were transferred to each healthy seedling of
1fe Brown.
42
After an overnight inoculation period, plants were sprayed
with dimethoate (0.15%).
The
inoculated seedlings were kept in an
insect-proof screenhouse for one month and symptoms were recorded for
virus infection.
2.8.2
Seed transmission
Seeds harvested from the fort y improved cowpea breeding
lines artificially infected with the following isolates -- 'ONNE',
liT 11', liT 16' and 'Zaria' (see 2.7) -- were sown in germination trays
containing steam-sterilized soil.
The seedlings were grown to the
second true leaf stage (about thirty days after sowing).
AlI the plants
were sprayed weekly with dimethoate (0.15%).
With the appearance of the first trifoliate leaves, the
seedlings were observed daily and checked individually for any viral
symptoms.
Seedborne infection was determined by symptoms on the primary
and first two trifoliate leaves.
Plant showing symptoms and the total number of plants esta-
blished were counted separately.
The virus identity was confirmed by
inoculating selected host plants and by serology.
2.9
Purification
The virus isolates were propagated in either V. unguiauZata
cv. Ife Brown or N. benthamiana and systemically infected leaves were
used for virus purification.
The first trifol iate leaves showing typical
mosaic symptoms were collected 10-15 days later and subjected to the
following purification procedure as described by Taiwo et aZ. (1982).
Four to five hundred grams of cowpea foliar tissues were
ground in a Waring blender in 0.5M phosphate buffer (pH 7.5) containing
1% sodium sulfite (w/v) and O.OlM EDTA and the sap filtered through four
layers of cheesecloth.
After adding 6.5% of n-buta~ol to the sap and
stirring overnight at 4°C, the mixture was clarified by centrifuging at
11,700 9 for five minutes in a Sorvall GSA rotor.
The aqueous phase was
collected and the virus precipitated by adding 7% (w/v) polyethylene gly-
col MW 8000 (PEG 8000).
After two hours of stirring at 4°c, the preci-
pitate was collected by low speed centrifugation for 15 minutes at
14,600 9 in the GSA rotor.
The pellet was resuspended in 25 ml of 0.02M
potassium phosphate buffer at pH 8.2 containing 0.1% 2-mercaptoethanol.
The suspension was stirred slowly overnight at 4°c.
Cesium chloride
(CsCl) was added to the suspension to make a 30% (w/w) CsCl solution.
This mixture was subjected to high speed centrifugation for 18 to 20
hours at 30,000 rpm in an
ultracentrifuge Beckman l5-50 using a SW-41
rotor.
After this first cesium chloride centrifugation, the viral
zone was removed by making a puncture in the bottom of the tube with a
needle.
This viral suspension was dialysed against 1,000 ml of phosphate
buffer (O.02M potassium phosphate pH 8.2) at 4°C with thre~ four chan-
ges.
Enough CsCl was added to the suspension to make the same solution
of CsCl as above, and a second high speed centrifugation was performed.
At the end of this second ultracentrifugation, the virus zone was removed
and dialysed again.
A highly purified viius preparation was obtained.
AlI the virus preparations were kept in a freezer after adding an equal
volume of glycerol.
2.10
Ultraviolet absorption spectrum of purified virus
Ultraviolet absorbance of the purified virus suspensions was
measured in a Beckman mode 1 25 spectrophotometer in the range of 230-
320 nm.
Absorbance values were not corrected for light scatterring.
Virus concentration was estimated by using an extinction coefficient of
2.4 at 260 nm (Taiwo et al., 1982).
2.11
Serological studies
2.11.1
Antiserum production
An antiserum to the Zaria isolate was prepared by intra-
muscular injections of an adult rabbit with freshly purified virus
44
emulsified with Freund's incomplete adjuvant (1:1, v/v).
The rabbit was
injected twice during the first week with about 0.8 mg of purified virus
on each occasion.
This was followed by one 0.5 mg injection and two
others of 0.5 mg at three and eight weeks after the first injection
respectively.
The rabbit was bled ten days after the first three injections
and two months later after the last intramuscular injection by making a
small incision with a sharp razor blade on the marginal ear vein and
collecting about 40 ml of blood.
Blood was left at room temperature for
thirty minutes to clot.
It was then kept at 4°e overnight after which
it was given a low speed centrifugation for five minutes at 3,020 9
(5534 rotor) to pellet the blood cells.
The serum was kept at -200 e.
2.11.2
SOS - immunodiffusion tests
Immunodiffusion tests were performed in agar plates
prepared according to Purcifull and Batchelor (1977).
The medium
consisted of 0.7% Noble agar, 1.0% sodium azide and 0.5% sodium dodecyl
sulfate (SOS).
The wells in the agar plates were made using a gel cutter
and the pattern consisted of six peripheral and one central one.
Wells
were 3.5 mm from edge to edge.
A modified gel-immunodiffusion method for flexuous virus
particles, as recommended by ToI in (1977), was also used.
In plastic
Petri-dishes were poured 14 ml of freshly prepared medium consisting of
0.6% Noble agar, 0.3% SOS and 1% sodium azide.
Antigens, either from
crude sap extracted with a mortar and pestle in a minimum amount of
distilled water or purified virus, were placed in the peripheral wells.
The antiserum was placed in the center weIl.
For some of the experi-
ments, the antiserum was first cross-absorbed with juice from healthy
plants to minimize the formation of non-specifie precipitation 1ines.
45
Plates were kept in a moist chamber at room temperature and
observed after 24 and 48 hours.
Antisera against 'DNNE ' and 'Zaria '
isolates were prepared at liTA.
The following antisera were obtained
from Dr. F.J. Morales (lI Centro Internacional de Agricultura Tropical 1,
ClAT, Cali, Colombia):
- bean common mosaic virus antiserum (BCMV-AS) (Florida
isolate)
bean yellow mosaic virus antiserum (BYMV-AS).
Antisera to BICMV (Florida isolate) and CAbMV (Kenyan, Nigerian and
Cyprus isolates) were obtained from Dr. M.A. Taiwo (University of Lagos,
Nigeria).
CHAPTER 3
RESULTS AND DISCUSSION
3. 1 Resul ts
3. 1. 1 Su rvey
Of the 140 field samples examined, 100 produced mosaic
symptoms on Ife Brown and 40 did not develop any symptom.
From the
infected plants, 40 had fllamentous particles and the other 60 had iso-
metric particles.
The 40 samples with flexuous particles were distri-
buted (Fig. 1) as follows:
Ibadan (Oyo State):
23 samples
Kano (Kano State):
5 samples
ONNE (Rivers State):
1 sample
Umudike (Imo State):
6 samples
Zari a (Kaduna State): 5 samples
Among these 40 samples, 16 produced mixed reactions (pot y-
virus + CuMV) which were collected at Ibadan (10 samples) and Umudike
(6 samples)
(Fig. 1).
47
During the survey, other viruses were identified in addition
to the potyviruses (CMeV, CYMV and CuMV).
CYMV was recovered in samp1es
from Oyo and Kwara States.
CMeV was detected in one samp1e
co11ected
near Funtua (Kaduna State).
CuMV was observed a1so in samp1es from
Ilorin (Kwara State) and Mokwa (Niger State)
(Fig. 1).
3.1.2
Identification of the iso1ates
From the 40 samp1es showing filamentous virus particles, the
fo110wing isolates were se1ected for further characterization.
The iso-
1ates 'ONNE' and 'Zaria' were co11ected from infected cowpeas
at
ONNE
and Zaria respective1y.
The fo110wing 1ist of iso1ates were from the
liTAis main site at Ibadan and had as original hosts:
- African yam bean (iso1ate lAT-S');
- Bambarra groundnut (iso1ate IBT-4');
- cowpeas (i so 1ates liT 11 l, '1 T 15' and '1 T 16 1 );
- wi1d cowpea (iso1ate 'WC I ).
Electron microscopie examination of crude juice from the
eight isolates revea1ed fi1amentous partic1es typica1 for potyviruses
(Figure 3).
A11 the iso1ates reacted positive1y in SDS agar gel diffusion
tests using an antiserum against the iso1ate 'ONNE' which served as a
reference iso1ate (Table 6).
Seven iso1ates were aphid transmissible in
non-persistent manner (with Aphis araaaivora Koch.) (see Table 13).
The grouping of the iso1ates (as B1CMV or CAbMV) was based
on the symptomato10gy on differential cowpea cultivars deve10ped by
Taiwo et al.
(1982).
The iso1ates 10NNE', 'AT-S', 'BT-4 1 ,
'IT 15' and
'WC' reacted on se1ected cowpea cultivars, 1ike B1CMV, whereas 'IT 11',
'IT 16' and 'Zaria' were considered to be CAbMV (Table 7).
Iso1ates were regu1ar1y indexed on N. gêutinosa L. and found
free of any contaminant cucumovirus.
The iso1ate ~T 11'was origina11y a
48
500 nrn..
Figure 3.
Electron micrographs of virus particles of CAbMV
A..
Isolate
'WC I
(leaf dip preparation)
stained with PTA
(2%)
(see section 2.4.1
for details) (X 50 000)
B.
Iso1ate 10NNE I
(purified virus)
stained with PTA (2%)
Magnification X 30 000
El ect ron microscope:
I/S i emens El mi skop 10211
49
TABLE 6.
Reactions of CAbMV comp1ex iso1ates to different antisera in
double immunodiffusion tests (serologica1 identification)
Antisera
1!
Iso1ates
!
r
AT-5
++
BT-4
++
1T 11 M
(+)
+
IT 15
++
IT 16
+
ONNE
++
wc (= wi1d cowpea)
Zari a
(+)
(+):
weak positive reaction
+:
positive reaction
++:
st rong pos i t i ve reac t ion
no reaction
(a):
immunodiffusion tests with sos
(b):
illlTlunodiffusion tests without SOS
TABLE 7.
Reactions of CAbMV and/or B1CMV isolates on selected differential cowpea cultivars
Isolates
Cowpea
1i nes
ONNE a
BT-4a
WC a
AT-5a
IT 15a
1T 11 b
Zari ab
IT 16b
TVu 196
Mo seVe
Mo, GVb
Mi 1d Mo
Mo
Mo
TVu 401
TVu 1582
Mo, Mt
Mo
Mo, Vb
Mo
(Mo)
TVu 1593
Mo
Mo
Mo
Mo
Mt
TVu 2657
(Mo)
Mo
(Mo)
(Mo)
Mo, GVb
Mo, GVb
Mo
TVu 2740
Mo
Mo, GVb
Mo, GVb
Mo
TVu 3273
(Mo)
(Mo)
Mi Id Mo,
GVb
Mo
Mo
TVu 3433
(Mo)
(Mo)
Mo, GVb
Mi Id Mo
Mo
GVb:
Green ve i nband i ng
Mt:
Mott 1i ng
Mi ld Mo:
Mild mosaic
( -) :
No symptoms
Mo:
Mosaic
a:
Isolates identified as BICMV
(Mo):
Mosaic with few plants infected
b:
Isolates identified as CAbMV
V1
Mo Sev.:
Severe mosaic
o
""'''''-~'''''''''"''''''''''''''''~'-''"''~.~~~''''''~:'~--'''''''~-'''''''"".~,!,~,,.,..... ,_._.,.,-....g ..... '>,...,--,,--''''........_ ..,.•.,., •..,''-~'''';~._..~".,.,..,"_.,...,...,..._."'~~-"~"""""""""''''
•. . . , - - - ..~....,...,...".-----~
... '"..-,~'.........---..."",.
51
mixture (potyvirus + CuMV).
The filamentous component was separated from
CuMV by using Cassia tOTa L. on which obvious necrotic local les ions
developed in few days.
C. tora was immune to the infection by CuMV.
3.1.3
Host range and symptoms
3.1.3.1
Locallesion
hosts
Chlorotic or necrotic local les ions were induced on the
following plant species:
FAMILY AMARANTHACEAE
- Gomphrena g7,obosa L.
The isolates 'ONNE', 'AT-S', 16T-4 1 ,
liT 16 1 and 'Zaria l
produced necrotic local les ions but the results were not always consis-
tent.
The other isolates (liT 111, liT 15 1 and IWC I) were not tested.
FAMILY CAESALPINACEAE
- Cas sia tora L.
The i so 1ates 1ONNE 1, liT 11', liT 15 1 and 1Za ria 1 gave nec ro-
tic local lesions.
liT 16 1 did not produce any symptom.
The other iso-
lates were not tested.
FAMILY CHENOPOVIACEAE
Chenopodium amarantiao7,or Coste & Reyn. and C. quinoa Wil Id.
AlI eight isolates produced chlorotic local lesions.
FAMILY LABIATAE
- Oaimum basi 7,iaum L.
Isolates IONNE', liT 111, liT 16 1 ,
IWC I and IZaria' induced
52
necrotic local lesions two weeks after the inoculation.
The other isola-
tes were not tested.
Only C. amaranticolor, C. quinoa and Cassia tora gave consis-
tently the same results.
The other plant species reacted in an erratic
manner.
3.1.3.2
Leguminous species with systemic reactions
Most of the isolates infected the following leguminous plants
causing various mosaic symptoms sometimes associated with necrotic local
lesions.
In some cases, systemic reactions induced mosaic, mottling and
veinbanding.
FAMILY LEGUMINOSAE
1. . Cyamopsis tetragonoloba (L.) Taub.
'ONNE' and 'WC' produced symptomless infection.
Isolates
'IT 11', liT 15', 'AT-S'and 'BT-4' induced mottling symptoms
but
'IT 16' and 'Zaria' did not infect this plant species.
2.
Glycine max (L.) Merr. (cvs. 'Bragg' and 'Davis')
Both cultivars infected by 'ONNE' reacted with mosaic and
occasionally
blistering symptoms.
The CAbMV isolates 'IT 16 1 and
'Zaria' infected only the cultivar 'Davis' producing mosaic symptoms.
The other isolates were not tested.
3.
Phaseolus lunatus L. (cv. TPL 250)
The isolate 'ONNE' provoked severe mosaic and mottling.
With
isolates lAT-S'and 'BT-4' the symptoms (mosaic) were mild.
The CAbMV
isolate 'IT 16' induced a severe mosaic with mottling.
53
4.
Phaseolus vulgaris L. (cvs. 'Black Turtle l and 'Pinto')
Isolates 'ONNE' and liT 16' induced mosaic on the cultivar
'Pinto'.
On 'Black Turtle', 'ONNE' produced necrotic local lesions
combined with mosaic.
'IT 16' induced
mosaic on the same cultivar.
Isolates 'WC' and 'Zaria' did not infect the cultivar 'Pinto'.
The
other isolates (liT 11', 'AT-5', 'BT-4' and liT 15') were not tested on
'Pinto' cultivar.
The CAbMV isolates 'IT 11' and 'Zaria' produced necro-
tic local '~esions on Black Turtle " . The isolates 'BT-4' and 'WC' induced
necrotic local lesions on the same cultivar (Figure 4).
The other iso-
lates 'IT 15' and 'AT-5' did not infect 'Black Turtle l .
5.
Vigna subterranea (Thouars) Verde.
Isolates 'ONNE', 'IT 16', 'AT-5', 'BT-4' produced more or
less severe mosaic and 'Zaria' isolate induced mild mosaic.
Mosaic
symptoms were more severe with 'BT-4' (blistering, leaf deformation and
veinbanding) (Figure 5).
Isolate 'AT-5' induced mosaic, leaf deforma-
tion and puckering (Figure 6).
Among the CAbMV isolates, 'IT 16' induced
severe mosaic with leaf deformation similar to the one developed by
'BT-4'.
The isolates 'IT 11', 'IT 15' and 'WC' were not tested.
3.1.3.3
Non-leguminous plants with systemic infection
FAMILY SOLANACEAE
a
- Niaotiana benthamiana Domin.
Isolates 10NNE', 'BT-4' and 'WC' induced mosaic and vein-
banding symptoms.
The CAbMV isolates 'IT 11' and 'Zaria' also induced
similar symptoms on N. benthamiana.
But this plant species was not
infected by isolates 'AT-5 1 ,
'IT 15' and liT 16'.
Subsequent tests on
Ife Brown as weIl as electron microscope examination gave negative
results.
54
Figure 4.
Necrotic local
lesions induced by isolate 'wei on Phaseolus
vulgaris L. cv. Black Turtle
LL:
1a ca l I e 5 i on 5
55
Figure 5.
Symptoms of mosaic, leaf deformation and veinbanding produced
by isolate 16T-4' on Vigna subterranea (Thouars) Verde.
(Vo
7326)
1:
Leaflets of infected plants
CP:
Leaflets of control plants
56
Figure 6.
Symptoms of mosaic and leaf deformation (puckering) induced
by isolate 'AT-S i on Vigna subterranea
(Thouars)
Verde.
(Vo 7326)
1:
Leaflets from infected plants
CP:
Leaflets from control plants
57
3.1.3.4
Non-hosts for both viruses
The following plant species did not develop any symptom with
the eight isolates.
No virus could be recorded in subsequent tests with
Ife Brown.
1.
FAMILY CHENOPOVIACEAE
i
i
- Spinacia oleraceae L.
i
1
1
2.
FAMILY CUCURBITACEAE
~
!!
1
- CUcumis sativus L. cv. Palmetto
1
- C. sativus L. cv. Marketmore
1
CUcurbita pepo
L.
t
3.
FAMILY LEGUMINOSAE
Arachis hypogaea L.
- Cajanus cajan Millsp.
- Canavalia ensiformis (L.) D.C.
Desmodium ovalifolium
Wall.
Lab lab purpureus L.
Phaseolus lunatus L. (cv. TPL 84A)
pisum sativum L. cv. Koroza
- Vicia faba L. cv. Compacta
4.
FAMILY SOLANACEAE
- Lycopersicon esculentum Mill. (cv. Roma)
Nicotiana glutinosa L.
N. megalosiphon Huerch. & Muell.
- N. rustica L.
To conclude this paragraph, we noticed that CAbMV isolates dif-
fered in symptoms severity on many leguminous and non-leguminous plants.
But their virulence in one plant species did not ne~essarily paraI leI that
in other species (Tables 8 and 9).
Vigna subterranea was found to be a
new susceptible test plant for CAbMV isolates.
:1!
58
TABLE 8.
Reactions of CAbMV isolates on selected susceptible plants
Isolates
Plants
1T 11
Zaria
1T 16
Vigna unguiauZata
1fe Brown
TVu 612
IT 82E-9
IT 82E-16
1T 820-975
TVx 3236-6-1
Niaotiana benthamiana
PhaseoZus vuZgaris cv.
Black Turtle
B-MO: Bright yellow mosaic
Vb:
Veinbanding
GMo:
Green mosaic
B-Mo severe: Severe bright yellow
Mo:
Mosaic
mosaic
NLL:
Necrotic local les ions
( -) :
No symptom
TABLE 9.
Reactions of B1CMV isolates on selected host plant species
Isolates
Plants
ONNE
BT-4
wc
AT-5
IT 15
Vigna unguiau Zata
1fe Brown
Mo
Mo
Mo
B-Mo
B-Mo
TVu 645
(Mo)
B-Mo
B-Mo
IT 82E-10
(Mo)
B-Mo
B-Mo
Niaotiana benthamiana
Mo
Mo
Mo
PhaseoZus vuZgaris cv.
Black Turtle
NLL (Mo)
NLL
NLL
Mo:
Mosaic
(Mo) :
Mosaic with few plants infected
NLL:
Necrotic local les ions
B-Mo:
Bright yellow mosaic
(-) :
No symptom
59
3.1.4
Reactions of CAbMV and/or B1CMV iso1ates on cowpea cultivars
germp1asm accessions and breeding 1ines
3.1.4.1
Reactions on cowpea germp1asm accessions
CAbMV and/or B1CMV iso1ates induced readi1y distinguishab1e
reactions on cultivar Ife Brown (Figures 7 and 8B).
B1CMV iso1ates can
be grouped into two categories according to symptoms produced on this
cowpea cultivar (Ife Brown).
B1CMV iso1ates 'ONNE, 'BT-4' and 'WC'
induced necrotic local les ions on inocu1ated primary 1eaves.
Systemic
reactions consisted in more or 1ess severe mosaic with puckering,
b1 istering of 1eaf1ets and stunting of infected plants.
Iso1ate 'BT-4 1
induced severe symptoms (b1istering and puckering) on different cowpea
cultivars and was the most virulent among the five B1CMV iso1ates.
The
second group inc1uded iso1ates IAT-5' and 'IT 15' which consistent1y
induced bright ye110w mosaic (no necrotic 1esions) on Ife Brown (Figure
8B).
The members of this 1ast category did not infect N. benthamiana.
Among CAbMV iso1ates we a1so distinguish two categories.
The first inc1uded liT 11 1 and 'Zaria' which expressed mi1d green mosaic
with veinbanding on 1eaves of cowpea Ife Brown.
No necrotic local
les ions were detected on primary inocu1ated 1eaves.
'IT 16'
represented
the second category with its characteristic bright ye110w mosaic with
occasiona1 veinbanding.
'IT 16' did not infect N. benthamiana.
This
iso1ate caused the more severe symptoms on plant species tested.
The
bright ye110w mosaic, induced by liT 16' on Ife Brown, was observed on
other cowpea cultivars (e.g. TVx 3236-6-1, see Figure 11).
Severa1 other cowpea germp1asm accessions were inocu1ated
with the CAbMV and B1CMV iso1ates.
Resu1ts (Table la)
indicated that,
many of the cowpea 1ines were susceptible to both viruses, but two
of them, TVu 401 and TVu 1948 showed high 1eve1s of resistance to a11
iso1ates of CAbMV and B1CMV.
TVu 1ines 222, 612, 645 and 1453 a1so
possess good 1eve1s of resistance to some iso1ates of both viruses
(Table la).
60
Figure 7.
Symptoms of bright yel10w mosaic (B) and common mosaic (A)
induced by isolates
'AT-5'
(B) and
IBT-4 1 (A) on Vigna
unguicuZata (L.) Wa 1p. cv. 1fe Brown
6 J
Figure 8.
Symptom of bright yellow mosaic induced by isolates lAT-S i on
Vigna unguiculata (L.) Walp. TVu 645 (A) and liT 15' on
V. unguiculata cv.
Ife Brown (B)
62
TABLE 10.
Reactions of CAbMV and B1CMV isolates on selected cowpea
germplasm accessions
Isolates
Cowpea
1i nes
1T 11
IT 16
Zaria
ONNE
IT 15
AT-5
BT-4
wc
TVu 21
+
+
+
TVu 128
+
+
+
+
+
TVu 201
+
+
+
+
TVu 222
+
+
TVu 345
NT
+
+
+
+
""\\
TVu 347
+
+
NLL
+
+
+
TVu 401
TVu 612
+
+
TVu 645
+
(+)
+
+
TVu 1453
(+)
TVu 1948
+:
Infected (Mosaic)
(+):
Few plants i nfected (Mosa i c)
-.
Not infected
NLL:
Necrotic local lesions
NT:
Not tested
f
~
1
,f
~
f
1
1
.
t
r
1
~:
f
63
TVu 128 was susceptible to five of our isolates (' IT 11',
'IT 16', 'BT-4', 'WC' and 'Zaria') (Figure 9).
However, it remained
symptomless after inoculation with isolates 'ONNE', 'AT-S'and 'IT 15'.
3.1.4.2
Reactions of isolates 'ONNE', 'IT 11', liT 16 1
and 'Zaria' on fort y cowpea breeding lines
On the basis of symptom development and effects on plant
growth 30 days after inoculation on the primary leaves, the reactions
of the fort y cowpea lines were distinguished as:
1.
resistant/moderately resistant (scores 1 and 1-2);
2.
moderately susceptible/susceptible (scores 2, 3 and
2-3);
3.
very susceptible (scores 3-4,4,4-5 and 5).
There was segregation. (S) when some of the plants showed
symptoms which differed in intensity from those exhibited by other
plants of the same line.
Results of the evaluation of 40 cowpea breeding 1ines (Table
11) showed that sources of resistance to CAbMV and/or B1CMV isolates
occurred among several cowpea lines.
Some of these lines had a broad
spect rum of res i stance to CAbMV and BI CMV.
Among these 1i nes:
1T 820-
885, IT 820-889, IT 82E-60 were resistant to four virus isolates (Table
Il) .
1n a sepa rate expe r i men t, the same cowpea 1i nes exh i b i ted good
levels of resistance to isolates 'AT-S', 'BT-4' 'IT 15' and 'WC' (Table
12) .
Cowpea line IT 820-752 was moderately susceptible to isolate.
liT 11' and remained symptomless with other isolates ('ONNE', 'WC',
'AT-S', 'IT 15', 'IT 16' and 'Zaria').
But the same line was severely
infected by 'BT-4'.
64
A
B
Figure 9.
5ymptoms of CAbMV iso1ates on Vigna unguieulata (L.) Wa1p.
TVu 128 (A and B)
A.
1 -
IONNE 1
B.
1 -
'ONNE '
2 -
1 1T16 1
2 -
'Zaria'
3 - liT 11'
3 -
liT 11'
4 - IBT-4 1
65
Figure 10.
Symptoms of CAbMV isolates
liT 16 1 (A),
'Zaria'
(8)
and isolate
10NNE I
(C) on the cowpea breeding line
IT 82D-709
66
Figure 11.
Symptoms of bright yel low mosaic and veinbanding induced
by the CAbMV isolate liT 16 1 on the cowpea breeding l ine
TVx 3236-6-1
67
1
TABLE 11.
Reactions of cowpea breeding 1ines and germplasm accessions
to four isolates of CAbMV and/or B1CMV
Isolates
Cowpea lines
ONNE
Zari a
1T 16
1T 11
IT 820-709 a
4 b
3
4
1T 820-713
3
2
3
1T 82E-9
4-5
1-2
4
2
'T 82E-16
2
2
3
2
IT 82E-18
3
2-3
3-4
2
IT 82E-32
2
2
3
IT 82E-60
1
1
1
IT 820-789
1 *
2
3
2
IT 820-812
S
1
2-3
IT 820-885
1
1
1
IT 820-889
1
1
IT 820-948
4
2-3
2-3
1
TVx 1948-01F a
2-3
2-3
3
1
IT 820-927
3
2
3-4
1
TVx 3236-6-1
3
1
4
1
IT 820-849
1
1
2
1
TVx 5881-016E
3
2-3 (S)
2-3 (S)
1
IT 820-380-5
2-3
2
3-4
1
FARVE 13
3
2-3
3
2
TVu 21
2-3
1-2
2
1
f
TVx 3442-027E
2-3
1-2
2
1
1
IT 810-1228-16
!
3
2
4
1
,
TVx 4659-03E
2
2
3
1
!
TVx 3236
2-3
l ,',
2
1 *
1
IT 820-975
3
1
4
1 *
IT 820-786
1-2 (S)
1
1 *
1
1 *
IT 820-752
t
1
1
1-2
IT 820-744
2-3
2
2
1
IT 820-716
1
2-3
2
1
!
1
1
68
a:
IT 820-709
Improved cowpea lines obtained at liTA with
(and other IT numbers)
IT for liTA
82 - for the year 1982
o
= for dry season
E
= for early season
709 = for plot number
TVx 3236 (and other TVx number) are cowpea crosses obtained at liTA.
b:
Oisease ratings were based on 1 to 5 scale with:
1.
No disease symptoms
2.
Mil d mosa i c
3.
Moderate mosaic
4.
Severe mosaic
5.
Severe mosaic with blistering and stunting
1-2, 2-3. 3-4 and 4-5:
were intermediate between two ratings.
S:
Seg regat ion
*
Symptomless but positive after back inoculation
There were differentia1 responses of the fort y cowpea 1ines
to the four iso1ates studied.
In addition, the severity of the symptoms
varied with the iso1ates.
This is an indication that there is variation
with virulence within the CAbMV and/or B1CMV iso1ates (Figure la).
We noticed that the breeding 1ines IT 820-752, IT 820-885,
IT 820-889 and IT 82E-60 were resistant to at least 6 of our virus iso-
1ates (Table 12).
Most of those 1ines had multiple resistance to severa1
funga1 and bacteria1 diseases in Nigeria (Sing et al., 1984).
3.1.5
Transmission studies
3.1.5.1
Aphid transmission
Most of the iso1ates were transmitted by Aphis craccivora
Koch in a non-persistent manner from cowpea to cowpea (cultivar Ife
Brown).
The Bambarra groundnut iso1ates (IBT-4 1 ) was transmitted by the
same aphid species from Vigna subterranea to Vigna unguiaulata cv. Ife
Brown).
The isolates 10NNE', 'IT 11', liT 15 1, liT 16 1 and 'Zaria l were
easi1y transmitted by this aphid.
On1y the iso1ate 'WCI was not trans-
mitted by Aphis craccivora after many unsuccessfu1 attempts.
The rates of aphid transmission are recorded in Table 13.
There was variation in aphid transmissibi1ity between the CAbMV and/or
B1CMV iso1ates.
3.1.5.2
Seed transmission of the iso1ates 10NNE I , 'IT 11',
'Zaria' and liT 16' in fort y cowpea breeding 1ines
The highest seed transmission (8.3%) was recorded with the
cowpea 1ine IT 82E-16 and the iso1ate 'IT 16', whi1e no seed transmission
was noticed with the same iso1ate in many other 1ines (Tables 14 and 15).
liT 16 1 was seedborne to the extent of 8.3%, 1.4%, 1.8% in IT 82E-16,
IT 820-975, IT 810-1228-10 respective1y.
The Zaria iso1ate was seed-
transmitted at the rates of 3.2% in IT 810-1228-16 and 1.6% in IT 820-786.
TABLE 12.
Selected sources of resistance (cowpea lines) to B1CMV and CAbMV (Nigerian isolates)
1so 1ates
Cowpea 1i nes
Remarks
ONNE
BT-4
wc
AT-5
IT 15
Il 11
1T 16
Zari a
- -
-
- -
- -
TVu LINES
- - - - -
a
TVu 222
R .
ss 0)
R
R
R
ss (2)
R
R
6/8
TVu 401
R
R
R
R
R
R
R
R
8/8
TVu 612
R
ss 0)
R
R
R
R
ss (2)
R
6/8
TVu 1453
R
ss (4)
R
R
R
R
R
R
718
TVu 1948
R
R
R
R
R
R
R
R
8/8
BREEVI NO LI NES
- - - - - - -
IT 820-752
R
SS (4)
R
R
R
MR
R
R
6/8
IT 820-885
R
NT
R
NT
R
R
R
R
6/6
IT 820-889
R
NT
R
NT
R
R
R
R
6/6
IT 82E-60
R
NT
R
NT
R
R
R
R
6/6
R:
Resistant
MR:
Moderately resistant
SS:
Susceptible (with corresponding scores or ratings)
NT:
Not tested
(a): Number of isolates to which this particular cowpea line exhibited resistance / total number
of isolates.
........
o
·~·'··"'__··~:~<"'··""·"'I~~'">('''-'i''''''~''·=.~V.'''x"(8'''''_'''';-'''''''~''··"''''''''''''_'''''''~''''''''''''~''''·''='''''':'''·''.''-_""_"~"''''_''''''''''''m~"
., ','P'" .'_,."",,' ,.,.''''-: {,""~-'':'"="9'-''''_''''fo/'"=~'''''''''''''''''-''''''i"'7''-~'''''~<!'''''''~''''t'''" __.":~,~,,,,.,...,,.*,,,,,,.,,,.... '~_·'<'.'''''v~;",·,.,,,,,,,,,,._=,,,,,,,,,,,,,,, __,,,,,,~.,,~,",,.,,,,_-.<,,...~,,~,,,,,,,·,,,.«.,,,"""!""".''''-*>'''~_~·_',",''-.'''''''''~'>'''''~·"""",_""_~~""""",,,,,,,,_~'''''_'''~'·,~m.,,,
...
"'K,.,,,,,,.,..."",._'''''''''_..._ ....
!,,!,,,"....,,,..~~..,""""".""'""'
......., . . . , . _ . " " " " ' _ ._ _
''''~,,_~_'''.,'' ..''.·.
71
TABLE 13.
Transmission of CAbMV and/or B1CMV isolates by Aphis cracci-
vora Koch ta V. unguiculata cv. Ife Brown l
ONNE
BT-4
WC
AT-5
IT 15
1T 11
Zari a
1T 16
Transmi ss ion
ta test
plants
2/l0a
1/10
0/24
2/27
3/16
3/20
2/17
3/20
Transmission
rate (%)
20 b
10
0
7.4
18.7
15
11.7
15
(a)
Numerator:
number of infected plants
Denominator:
number of inoculated plants
(b)
Aphid transmission rate in percentage
l
At least 10 plants were used for each isolate and 10 aphids were placed
on each plant.
The aphids were a1lowed an acquisition period of appro-
ximate1y one minute.
Inoculation period was approximately 16 hours.
73
TABLE 14 (continued)
ONNE
ZARIA
IT 16
1T 11
1T 810-1151
0/56
0/75
0/52
(0/9)b
IT 810-1137
0/64
0/83
(0/14)b
NT
IT 810-1064
0/30
0/64
0/80
(0/16)b
IT 810-1032
0/41
(0/3)b
NT
(0/24)b
IT 810-1007
(0/7) b
0/87
0/111
0/63
IT 810-985
(0/23)b
(0/26)b
(0/19)b
NT
IT 810-1228-10
0/62
0/36
0/52
NT
IT 810-1228-13
0/80
0/63
0/74
(0/13)b
IT 810-1228-14
0/39
0/32
(0/19)b
NT
(a)
Numerator: number of plants infected
1
1
Oénominator: total number of plants (= equal or more than 30)
1
(b)
Number of plants too low to allow conclusion
1
(c)
Seed transmission rate (percentage)
11
1
NT:
Not tested (seeds lost)
1
,f
1
1
1
1
!
,i
1
~
\\
[
74
(.
1
,
!
1
1
1
TABLE 15.
Seed transmission rates in selected cowpea breeding lines
inoculated with isolates 'ONNE',
1
'Zaria' , liT 16' and
liT 11' (data fram Table 14)
f
f
Isolates
f
Cowpea
ONNE
Zari a
IT 16
IT 11
1i nes
B
B
B
A
A
A
B
A
1
(%)
(%)
(%)
(%)
t
IT 82E-16
0/61
0
0/86
0
12/143
8.3
0/26
0
!
IT 82E-32
0/113
0
1/61
1.6
0/108
0
3/47
6.4
IT 810-1228-16
0/41
0
2/61
3.2
NT
NT
TVx 3236
0/106
0
0/84
0
0/103
0
2/58
3.4
IT 820-975
0/63
0
0/40
0
1173
1.4
0/6
0
IT 820-786
0/65
0
2/139
1.4
0/57
0
0/96
0
FARVE 13
0/18
0
0/6
0
0/25
0
3/38
7.8
A:
Number of infected plants / total number of plants
B:
Transmi ss i on rate (%)
NT: Not tested
75
Finally liT 111 had seed-transmission rates of 7.8% in FARVE 13,6.4% in
IT 82E-32 and 3.4% in TVx 3236.
The isolate 'ONNE' was not seed-
transmitted in any of the 1ines tested.
The followind cowpea lines
IT 820-786, IT 820-975 and TVx 3236 which were symptomless (see Table
Il) transmitted virus through seeds (Tables 14 and 15).
ln general, the rates of seed transmission of the isolates
seemed rather low.
However this is enough to represent high risks for
potential outbreaks.
In addition, there was variation in transmission
of the isolates tested through the seeds of infected cowpea lines (Table
15) .
3.1.6
Purification of the isolates
Isolates liT Il', 'ONNE', IWC' and 'Zaria' were purified from
N. benthamiana as weIl as from V. unguicuZata cv. Ife Brown by the proce-
dure described in section 2.9.
Isolates liT 16 1 and 'BT-4 1 were purified
fram cowpeas.
Virus preparations obtained after two cycles of centrifuga-
tion in CsCl showed only one major diffuse band at 20 mm from the botton
of the tube.
Electron microscope examination of purified virus revealed
numerous filamentous particles (Figure 3B).
Virus yields varied from
0.2 mg to 0.8 mg per hundred grams of foliar tissue.
3.1.7
SOS-immunodiffusion tests
SOS-immunidiffusion test, as recommended by Tolin (1977) was
used during the serological studies of CAbMV and/or B1CMV isolates.
Consistent results were
obtained using 0.6% agar, 0.3% sos and 1% NaN3
but the plates were difficult to handle.
Therefore, we preferred the
medium consisting of 0.7% Noble agar (with 1% NaN3 and 0.5% SOS) which
was used throughout this study.
Antiserum prepared against the 'Zaria' isolate reacted to
aIl isolates of B1CMV and CAbMV.
But the intensity of the reaction
varied with isolates.
No visible spurs have been observed between
isolates of both viruses.
76
Virus isolates 'ONNE', 'AT-5 1 ,
'BT-4' and 'WC' consistently
reacted with the 'ONNE' antiserum.
These isolates were similar or iden-
tical to BlOW.
In aIl tests, 'IT 15' formed strong precipitin lines
with the same antiserum.
Antiserum against the 'ONNE' isolate had a
titer of 1/1024 in micropecipitin test (Thottappilly, 1984) and it
reacted with three isolates ('IT 11', 'IT 16' and 'Zaria') belonging
to the CAbMV group.
AlI isolates reacted positively with antisera against BCMV .
and B1CMV (Kenya, Nigeria and Florida isolates) (Table 16).
The results obtained in agar gel diffusion tests were also
confirmed by the direct double antibody sandwich ELISA technique.
Iso-
lates 'ONNE', liT 11', 'IT 15 1 ,
'IT 16', 'WC' and 'Zaria' gave positive
reactions with ELISA using the antiserum against the rONNE' isolate.
AlI these findings revealed immunological relationships between the
isolates under study.
They share some common antigenic properties.
3.2
Discussion
The use of differential cowpea cultivars to distinguish
CAbMV from B1CMV was suggested by Taiwo et al.
(1982).
Some of these
cowpea cultivars were used during the present studies to classify our
isolates as B1CMV or CAbMV.
However,this biological grouping was
considered complementary to other methods such as host range studies
and serology.
Virus isolates 'ONNE', 'AT-S', 'BT-4', 'IT 15' and 'WC'
resembled B1CMV in terms of reaction on selected cowpea cultivars whereas
'IT 11 l, 'IT 16' and 'Zaria' can be considered to be more closely related
to CAbMV.
The identification of these isolates was supported by their
reactions on selected cowpea cultivars, host range studies as weIl as
serological studies.
77
TABLE 16.
Reactions of the B1CMV and/or CAbMV iso1ates to homo1ogous
and hetero1ogous antisera and to antisera of BCMV and BYMV
in SDS-immunodiffusion tests
Antisera
Iso1ates
CAbMV
ONNE
Zari a
Nigeria
Kenya
F10rida
BCMV
BYMV
CYPRUS
ONNE
++
++
++
+
++
+
BT-4
+
+
++
+
++
+
WC
++
+
++
+
+
+
AT-5
+
++
++
+
+
+
1T 15
++
++
++
+
++
+
Zari a
+
++
+
+
+
+
1T 11
+
++
+
+
+
+
1T 16
+
+
++
+
+
+
+
Positive reaction
(+)
Weak positive reaction
++
Strong positive reaction
Negative reaction
78
Some difficulties were noticed during the identification of
the virus isolates.
First, the expression of the symptoms sometimes
depended on the age of the plants and the environmental factors.
Second-
ly, segregation reactions observed among the cowpea plants complicated
the interpretation of the results.
In fact, many cowpea lines gave mixed
reactions showing a range of symptoms suggesting that there was genetic
variation within those cowpea germplasm accessions and breeding lines.
This could be used for genotype selection.
Similar heterogeneous reac-
tions were noticed by other authors (Ladipo and Allen, 1979a;
Pio-
Ribeiro and Kuhn, 1980;
Taiwo, 1978).
But, in general, the distinction
of CAbMV from B1CMV, based on differential cowpea cultivars (Table 7),
is val id.
The vi rus isolates 'AT-5 1 , 'BT-4' (B1CMV) and' IT 111 and
IZaria l (CAbMV) fit weIl the system suggested by Taiwo et al.
(1982).
The virus isolates 'WC', liT 15', 'ONNE' and liT 16 1 were difficult
to classify in one group.
These results suggest that the distinction
between CAbMV and B1CMV made in this work needs further studies.
In
addition,no reciprocal SDS-immunodiffusion tests were done,
there-
fore we preferred to consider B1CMV isolates (IONNE', 'BT-4 I ,
lAT-S',
liT 15' and 'WC I ) as CAbMV isolates.
The problem of mixed infections (CAbMV and CuMV) represented
another complication during the identification of the isolates.
About
40% of the field samples contained CuMV.
Mixed infections with CuMV
is important because of the synergistic reactions often produced in the
presence of CAbMV or B1CMV.
Sorne reports indicated that N. glutinosa was infected by
CAbMV (Kaiser and Mossahebi, 1975;
Ladipo, 1976) but we observed that
this plant became systemically infected only when CuMV was present in
the inoculum.
Therefore N. glutinosa is not a susceptible plant for
CAbMV even after repeated attempts to transmit this virus.
Previous publications indicated that Glycine max, Gomphrena
globosa and Pisum sativum were diagnostic plant species for CAbMV (Bock
and Conti, 1974;
Lovisolo and Conti, 1966).
In the present studies,
the first two plants were infected in an erratic manner.
No infection
79
occurred when Pisum sativum (cv. Koroza) was inocu1ated with our virus
iso1ates.
CUaumis sativus and CUaurbita pepo were found systemica11y
infected by CAbMV (Loviso10 and Conti, 1966).
None of these plants showed
symptoms with the CAbMV iso1ates.
Loviso10 and Conti (1966) described the
local reaction of Oaimum basiZiaum as of diagnostic value for CAbMV.
This plant species gave necrotic local les ions with our isolates in an
erratic manner.
The expression of this local reaction seemed to be
related to environmental factors.
This could explain the conflicting
results reported in the literature.
The isolates from African yam bean ('AT-S') and from Bambarra
groundnut ('BT-4 I ) were identified as CAbMV.
This is the first report of
natural infection of these crops by CAbMV.
In addition, Bambarra ground-
nut (Vigna subterranea) was a new susceptible plant found for this virus
in our host range study.
CAbMV was also detected in infected wild cow-
peas (isolate IWC I).
The CAbMV isolates infected two other wild cowpeas
(data not shown) when these were mechanically inoculated.
These findings
may have sorne epidemiological implications.
Since these leguminous plant
species could harbor CAbMV they may constitute carryover hosts between
seasons for this virus.
Niaotiana benthamiana was reported as a susceptible host
plant for CAbMV (Chang and Kuo, 1983;
Thottappilly and Rossel, 1984) but
we discovered that this solanaceous species was not infected by aIl CAbMV
isolates.
Therefore, the use of this plant species in host range studies
of CAbMV should take into account this new information.
The virus isolates identified as CAbHV exhibited variabil ity
on the basis of their symptoms (type and severity) on some host plant
species.
The first group of CAbMV isolates:
lAT-5 1 , 'IT 15' and liT 16'
induced severe bright yellow mosaic on susceptible cowpeas.
Variabil ity
in virulence was noticed with the second group of isolates including
'ONNE', 'BT-4' and IWC I •
Symptoms from these isolates on cowpea Ife
Brown consisted of more or less severe mosaic with
variable degrees of
dark green and yellow patches sornetimes accompanied by blistering and
puckering.
The isolate IBT-4 1 was the most severe among the three
previously cited CAbHV isolates.
And the third group with isolates
80
liT Il' and 'Zaria' produced mild green mosaic with veinbanding on
various cowpea cultivars.
However,
i sol ate
'1 TIl'
seemed
more virulent than 'Zaria'.
Similar variability in virulence was also
reported by others (Bock, 1973;
Taiwo et al., 1982).
In addition, iso-
lates lAT-5 1 ,
'IT 15' and 'IT 16' could not infect N. benthamiana while
'BT-4', liT 11', 'ONNE', 'WC' and 'Zaria' induced mosaic of variable
severity on this plant.
The variabil ity among the CAbMV isolates has some implications
for the breeding program at liTA.
While screening for resistance to
CAbMV, it would be safer to use a set of representative isolates of this
virus.
The danger of the use of one type of isolate in screening studies
is illustrated by the fact that the cowpea line TVu 128 was previously
reported as a good virus resistance donor because it was resistant to
six different cowpea viruses that are prevalent in Nigeria (Rossel and
Huttinga, 1983).
The virus isolate 'ONNE', which was extensively used
in screening studies for CAbMV at liTA, could not infect this cowpea
1ine.
In addition, IT 82E-IO was also reported to be resistant to CAbMV,
but the virus isolate 'IT 11' was recovered from this cowpea 1ine.
Most
of the isolates infected the two cowpea lines (TVu 128 and IT 82E-10).
Therefore, we suggest for screening studies the use of isolates IBT-4',
liT 11', 'IT 16' and 'ONNE'.
It is thus necessary to continuously
search for new sources of resistance and to survey for the prevalence
of new CAbMV isolates.
One important aspect of this work concerned the evaluation of
cowpea germplasm accessions and breeding 1ines for their resistance to
CAbMV.
The results
indicated
that
germplasm accessions
TVu
401
and TVu 1948 could be considered as good virus resistance donors since
they expressed high levels of resistance to both viruses.
These cowpea
lines were previously reported as resistant to a Nigerian isolate of
CAbMV (Ladipo and Allen, 1979a;
Taiwo, 1978).
TVu 1948 combines
resistance to CAbMV with relatively broad spectrum resistance to SBMV
(Ladipo and Allen, 1979b) and CVMV (Williams, 1977b).
TVu 1948 also
exhibits resistance to A. araaaivora (liTA, 1978).
81
1
Preliminary results showed that TVu lines 222,612,645 and
1453 possess relatively good levels of resistance to some of the isolates
!
(Table 10).
TVu 645 was found to possess resistance to some isolates of
i
CAbMV and CYMV (Williams, 1977a, b).
TVu 612 was also reported to have
resistance to certain isolates of CAbMV and SBMV (Ladipo and Allen.
1979b).
In present studies TVu 612 was infected by liT 16 1 and 'BT-4 1
1
and TVu 645 was susceptible to four of our isolates (Table 10).
TVu 128
previously reported as good virus resistance source (Rossel and Huttinga.
1
1983) because it was resistant to six cowpea viruses including CAbMV was
1
susceptible to five isolates and it remained symptomless to three other
1
CAbMV isolates.
!
f
AlI these findings demonstrate that sources of combined virus
resistance are available for incorporation in advanced cowpea breeding
lines.
These sources of resistance can allow for achieving the major
objective of the 'Grain Legume Improvement Program l at liTA in its
efforts to develop multiple disease resistant cowpea varieties
(Wi 11 iams. 1977a).
Among the advanced breeding 1ines evaluated for their resis-
tance to CAbMV. IT 820-885. IT 820-889 and IT 82E-60 were resistant to
six of our isolates (Table 12).
In previous work. the breeding lines
IT 82E-IO and IT 82E-16 were found to possess good levels of resistance
to isolate 'ONNE ' (Singh et al .• 1983).
Only the resistance of IT 82E-l0
to 'ONNE ' was confirmed in this study but
cowpea 1ine
IT 82E-16
was
susceptible to the same isolate.
In addition, this line gave the high-
est rate of seed transmission with isolate 'IT 16 1 •
Although the rates of seed transmission were rather low
(Table 15). they were high enough to permit seedborne infection to
develop into epidemic proportions.
There was variation in seed trans-
mission rates of four of B1CMV and/or CAbMV isolates.
These results
confirm findings from other authors that cowpea cultivars influenced
greatly rates of seed-borne infection of CAbMV and/or B1CMV (Aboul-Ata
et al., 1982;
Behncken and Maleevsky, 1977;
Fischer and Lockhart.
1976a;
Ladipo, 1976;
Lovisol0 and Conti. 1966).
In addition. the
82
aphid vectors are present throughout the year and initial seed infection
rate less than 1% could result in significant spread of CAbMV.
Seed
transmission of CAbMV has important epidemiological consequences, espe-
cially in international
transfers of improved cowpea varieties.
There-
fore efforts must be directed towards producing virus-free seeds.
A
selection for resistant cowpea lines to locally occurring CAbMV isolates
should help to reduce the incidence of this virus where cowpeas are
intensively grown.
Some of the cowpea breeding 1ines were infected but they
remained symptomless (e.g. IT 82E-32, TVx 3236) (see Table 11) and these
virus tolerant lines could represent natural sources of inoculum in the
cowpea fields (Table 15).
These results suppo;t the view that it is
more useful to breed cowpea for resistance to infection with CAbMV than
for tolerance.
Apparently non-transmitting genotypes may in fact
transmit virus through seeds with a very low incidence
not
detec-
table by visual observations.
Screening based on visual symptomatology
is not sufficient to identify one cowpea variety as resistant to one
particular virus isolate.
In the present studies we used back inocula-
tions on susceptible cultivars associated with electron microscopy and
sometimes serology tests for promising cowpea 1ines like those reported
in Table 12.
Very simple, sensitive, rapid and inexpensive tests for
detection of CAbMV in cowpea seed-lots or during screening tests could
be of valuable help.
One of these promising methods which can detect as
low as two nanograms/mL of virus could be the 'dot-immunobinding assay'
on nitrocellulose (Parent et aZ., 1985).
The Bambarra groundnut isolate ('BT-4') was transmitted by
Aphis c~ccivo~ from Vigna subter~ea to Vigna unguicuZata cv. Ife
Brown. This finding may have some epidemiological implications in the
mixed cropping systems (Bambarra groundnut and cowpeas) or if the two
leguminous crops are grown in the same area.
But isolate 'WC' was not
transmitted by this aphid species.
This was somewhat surprising since
we expected an easy aphid transmission of that isolate from wild cowpeas
to cowpea crops.
These wild cowpeas may constitute carryover hosts for
CAbMV between seasons.
The poor transmissibility of isolate 'WC' by
Aphis c~ccivora needs further study including other aphid species.
Clarification of extracts containing CAbMV was currently
performed with 8% of n-butanol (Bock, 1973;
Lima et al., 1979;
Taiwo
et al., 1982).
That concentration of n-butanol produced sI ight greyish
precipitates during the purification process.
The maximum concentration
of n-butanol used during this study was 6.5% which yielded better results.
It was better to purify isolates 'ONNE', 'BT-4', 'WC', liT 11' and
'Zaria' from N. benthamiana because high concentrations of virus were
reached in this plant.
For the other isolates (' IT 15', liT 16' and
'AT-S') any susceptible cowpea cultivar allowing high concentrations
of virus can be used for this purpose.
Viruses of the CAbMV isolates were not easily detected in
crude juice of infected cowpeas by means of SDS-immunodiffusion tests.
But consistent positive reactions were obtained by using N. benthamiana
as the source of virus.
Unfortunately, aIl isolates could not infect
this solanaceous plant.
ln non-reciprocal SDS-immunodiffusion tests using antiserum
to B1CMV, Lima et al.
(1979) demonstrated that B1CMV and CAbMV are two
distinct viruses.
Taiwo and Gonsalves (1982) using different serologi-
cal tests, showed that B1CMV and CAbMV are two related but distinct
viruses.
Results of SDS-immunodiffusion tests indicated that our virus
isolates belong to the same serological group since there was no visible
spur formation between the two groups of isolates.
Immunological rela-
tionships of isolates of CAbMV and B1CMV to other potyviruses have been
reported in the literature (Bock, 1973;
lima et al., 1973;
lovisolo
and Conti, 1966).
In the present studies, aIl isolates reacted positi-
vely with a BCMV antiserum, but not with a BYMV antiserum.
Although
aIl isolates reacted positively with a BCMV antiserum they cannot be
considered as BCMV because of differences in their host ranges.
BCMV
infected Cassia tara, inducing local and systemic reactions, while
CAbMV usually produced local lesions.
The negative results obtained with BYMV and CAbMV (CYPRUS
isolate) antisera are probably due to poor antigenic properties.
The
non-availability of homologous antigens (quarantine restrictions)
excluded further experimentation.
84
Areas of research needs
Further research should be directed to the following aspects:
1.
Production of antisera to other isolates.
2.
Comparative serological studies to establish relationships
between isolates.
3.
Study of extent of variability with CAbMV (and B1CMV)
isolates.
4.
Determination of stability of known sources of resistance
to CAbMV.
3.3
Conclusion
This study was undertaken to determine the occurrence and
variability of CAbMV in Nigeria and its implications for the cowpea gene-
tic improvernent program at liTA.·
Eight field tissues samples from cowpeas in Nigeria, con-
taining filamentous virus particles, were identified as CAbMV.
The iden-
tification of these isolates was supported by their reactions on selected
cowpea cultivars, host range studies as weIl as serological and electron
microscopie examination.
The incidence of CAbMV is important in each
ecoJogicaJ zone (forest and savanna).
It is widespread in Nigeria
since this virus was observed from Ibadan to Zaria.
ln host range studies, Vigna subterranea
was found to be a
new susceptible plant species for CAbMV.
The susceptibil ity of Nicotiana
glutinosa to CAbMV was not confirmed.
In addition, Cucumis sativus
and Cuaurbita pepo were not infected by CAbMV.
Ocimum basilicum gave
necrotic local lesions with our isolates in an erratic manner.
Results
of the host range studies suggest some biological groupings.
Virus
isolates of CAbMV can be divided into three groups according to the
symptoms induced on sorne selected host plant species (cowpea lines,
N.
benthamiana and phaseolus vulgaris cv. Black Turtle).
The
85
first
group
of
isolates
'AT-S',
'IT 15'
and
'IT 16'
induced
bright
yellow
mosaic
on
susceptible
cowpeas.
Isolates
'BT-4',
10NNE I and 'WC' of the second group produced mosaic with variable inten-
sities of dark green and yellow patches, sometimes accompanied by
blistering and puckering on cowpea Ife Brown.
Isolate 'BT-4' was the
most severe and virulent one.
The third group of isolates included
'IT 111 and 'Zaria' which produced mild green mosaic with veinbanding
on different cowpea cultivars.
Isolate 'IT 11' seems more virulent than
'Zaria'.
N. benthamiana was immune to artificial infection by lAT-5 1 ,
'IT 15' and 'IT 16' but the same plant species produced mosaic when
inoculated with isolates 'BT-4', 'ONNE', 'WC' and 'Zaria'.
There was
natural variability among CAbMV isolates according to their host range
and symptomatology.
The variability in virulence has important implications for
the Grain Legume Improvement Program at liTA.
First, in the screening
program for resistance to CAbMV, it would be better to use a set of
representative isolates of this virus in separate tests for each isolate.
We suggest the use of isolates 'BT-4', 'ONNE', 'IT 11' and liT 16'.
Secondly, it would be important to intensify the search for
sources
of resistance
to
CAbMV
and
to
look for new isolates
of
this
virus.
The cowpea germplasm accessions TVu 401 and TVu 1948, evaluated
for their resistance to our CAbMV isolates, can be considered as good
virus resistance sources.
The other cowpea lines identified as resis-
tant to either one or more isolates of CAbMV are TVu 222, 612 and 1453.
These findings demonstrate that sources of virus resistance are available
for incorporation in cowpea lines.
The advanced breeding lines, IT 82D-
752, IT 82D-885, IT 82D-889 and IT 82E-60 were found resistant to six of
our CAbMV isolates.
Differences between cowpea accessions were observed in the
rates of seed infection.
Symptomless plants also transmitted virus
through seeds.
These results support the view that it is more useful
to breed cowpeas for resistance to infection with CAbMV than for tole-
rance.
Mixed infections (CuMV and CAbMV) were often 'observed during
our surveys.
CuMV is widespread in cowpeas in Nigeria.
This virus is
86
transmitted through seeds, and in association with CAbMV it frequently
produces synergistic reactions.
Seed transmission of CAbMV and CuMV has
important epidemiological consequences, especially in international
transfers of improved cowpea varieties.
Therefore it is important to
intensify efforts towards the use of cowpea accessions with resistance
to virus transmission through seeds or the use of virus-free seeds.
CAbMV was recovered in Bambarra groundnut and African yam
bean.
The same virus was detected in wild cowpeas.
All these legumi-
nous plants probably constitute carryover hosts between seasons for
CAbMV.
Antisera prepared against 'ONNE ' and 'Zaria' and two
other isolates of the same virus (Kenya, Nigeria) reacted with all
CAbMV isolates but the intensity of the reaction varied.
No visible
spurs have been observed between isolates.
SDS-immunodiffusion tests
revealed that all virus isolates belong to the same serological group.
The results from agar gel diffusion tests were also confirmed by the
direct double antibody sandwich ELISA.
AIl these findings revealed
close immunological relationships between our CAbMV isolates.
Previous reports on potyvirus(es) in cowpeas in Nigeria
were essentially dealing with CAbMV.
Our results indicate that in addi-
tion to CAbMV, BICMV occurs in this country, but this needs to be
further studied.
LITERATURE CITED
ABOUL-ATA, A.E., ALLEN, D.J., THOTTAPILLY, G. and ROSSEL, H.W.
1982.
Variation in rate of seed transmission of cowpea aphid-
borne mosaic virus in cowpea.
Trop. Grain Leg. Bull. ~: 2-7.
ALLEN, D.J., ANNO-NYAKO, F.O., OCHIENG, R.S. and RATINAM, M.
1981.
Beetle transmission of cowpea mottle and southern bean mosaic
viruses in West Africa.
Trop. Agric. (Trinidad) 58: 171-175.
ANDERSON, C.W.
1955a
Vigna and Crotalana vi ruses in Florida.
Il. Notations
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