i* COWPEA WIRUSES IN SENEGAL, WEST AFRICA: ...
i*
COWPEA WIRUSES IN SENEGAL, WEST AFRICA:
IDENTITIES, DISTRIBUTION,
SEED-TRANSMISSION, AND SOURCES OF GENETIC RESISTANCE
M. Ndiaye, M. Bashir, X.E. Keller and R.O. Hampton
First author: Institut Senegalais de Recherches Agricoles / CNRA
de Bambey B.P. 53, Bambey, Senegal.
Second author: formerly
Department of Botany and Plant Pathology, Oregon State
University; nowNationa1 Agricultural Research Center, Islamabad,
Pakistan.
Third and fourth authors: U.S. Department of
Agriculture, Agricultural Research Service, Department of Botany
and Plant Pathology, Oregon State University, Corvallis OR
97331-2902.
Contribution of the USDA Agricultural Research Service in
cooperation with the Department of Botany and Plant Pathology,
Agricultural Experiment Station, Oregon State University,
Corvallis.
Technical paper 10177, Oregon Agricultural Experiment
Station.
This research was partially supported by the Bean/Cowpea Collabora-
tive Research Support Program of the United States Agency for
International Development, Grant No. DAN-131D-SS-6008-00.
Mention of a trademark or proprietary product does not constitute
a guarantee or warranty of the product by the U.S. Department of
Agriculture and does not imply its approval to the exclusion of
other products that also may be suitable.
ABSTRACT
Ndiaye, M., Bashir, M., Keller, K., and Hampton, R. 0.
1993 ?
Cowpea viruses in Senegal, West Africa:
Identities, distribution,
seed-transmission,
and sources of genetic resistance.
1
Viral diseases of cowpea in Senegal were surveyed during the
2
rainy seasons of 1990 and 1991.
Sixty-six viral-symptomatic plant
3
samples from five cowpea production areas were assayed for seven
4
viruses by DAC- or DAS-ELISA.
The following four recognized
5
viruses were detected, a11 of which are seed-transmissible in Vicma
6
unauiculata:
cowpea aphid-borne mosaic potyvirus (CABMV) (34/66),
7
cowpea
mottle carmovirus
(CPMoV) (2/66), cowpea severe mosaic
:
. .
)
i’
*
!p
?? ? ?
? ? ? ? ? ? ?
?
?
?
?
?
?
??
? ? ? ? ? ?
c
comovirus
(CSMV)(1/66), and
sou,thern bean mosaic sobemovirus
(SBMV)(:L/66).
In addition to these four viruses, variants of an
unknown potyvirus were detected in 21 of the 66 samples by use of
potyvirus-selective monoclonal antibodies (Agdia PTY and BCMV II-
5
197).
These potyvirus variants occurred principally in new
6
improved CABMV-resistant cowpea genotypes,
and their combin'ed
7
incidence was exceeded in plant samples only by CABMV.
Isolates of
8
the unknown potyvirus were seed-borne in Senegal cowpea lines and
9
were efficiently transmitted non-persistently by the cowpea aphid,
10
Aphis craccivora.
Selected seed-borne isolates of this potyuirus
11
were distinguishable principally by differentially resistantcowpea
x2
genotypes and by either weak (isolate Vl-1) or strong (isolate V17-
13
14)
reactions to
potyvirus-selective
monoclonal
antibodies.
14
Thirty-five
selected cowpea genotypes were tested as possible
15
sources of resistance to the unknown potyviruses.
Of these, six
16
(TVU-401, TW-408P2, TVU-1000, TW-1016-1, TW-1582, and White
17
Acre-BVR) were resistant to a11 isolates of the potyvirus.
These
X8
genotypes have been
included in the existing Senegal cowpea
19
breeding program for development of virus-resistant cultivars.
20
--
-
21
Cowpea [Vigna unguiculata (L.) Walp.] is second in importance
22
only to groundnut, among Senegal legume crops.
Senegal cowpeas are
23
annually grown on some 63,000 hectares, with an annual production
Plant Disease
Ndiaye, et a1
Page 2
3
1
of 18,C~OO,OOO kg.
Doubling the cowpea yield there should be
2
readily achievable, since the average yield of 280 kg/ha represents
3
only 14 to 28% of the 1,000 to 2,000 kg/ha yields in experimental
4
fields, in Senegal (19).
Several factors contribute to low yields
5
of cowpea in Senegal, and viral diseases are considered a major
'6
limitation.
7
Among cowpea viruses reported in West Africa (13, 15, 17, 1.9,
8
22) I cowpea yellow mosaic comovirus (CPYMV) and cowpea aphid-borne
9
mosaic
potyvirus
(CABMV)
are
considered
economically
most
10
important.
Other viruses known to occur in West Africa include
11
cowpea :mottle carmovirus (CPMoV) (1, 18) and southern bean mosa,ic
12
sobemovirus (SBMV) (7, 14).
Cowpea mild mottle carlavirus (CMMV),
13
cowpea
severe
mosaic
comovirus
(CSMV), and cucumber mosaic
14
cucumovirus (CMV) had been previously detected in seed lots from
15
Burkina Faso, Nigeria, Senegal, and Ghana, respectively (8).
16
Seed-borne viruses were considered a major constraint to
17
cowpea yield in Senegal farm fields (Gaikwad, D. G., unpublished
18
results).
Seed-borne viruses are especially destructive because
19
emerging plants are exposed early to seed-borne inoculum that is
20
acquired and progressively spread by insect vectors, particularly
21
by aphid and beetle species.
Two cowpea breeding lines, IS86-275N
22
(released in 1992 as cvMouride) and IS86-283-15N had been recently
23
developed to increase sustainable cowpea production in Senegal.
Plant Disease
Ndiaye, et a1
Page 3
.A.,
‘,.. , .; :,.
>..Oh
:
<i
_.---.~---.
--.
4
These lines were resistant to CABMV, bacterial blight (Xanthomonas
campestzis p v
vignicola) ,
storage
weevil
(Callosobruchus
maculatxs),
striga (Striga gesnerioides), and drought (19).
However, viral diseases in field trials of these lines in 1989-90
suggested the incidence of unrecognized indigenous viruses or
perhaps undescribed pathotypes of CABMV.
The present study was
conducted to isolate and partially characterize these viruses and
to identify resistant cowpea genotypes for use in developing virus-
resistant cultivars (16).
10
Materials and IMethods
11
Field survey and collection of viral isolates.
During the
12
rainy seasons of 1990 and 1991 fields were surveyed for viral
13
diseases in the five cowpea production areas of Senegal.
A total
14
of 66 samples was collected from viral symptomatic plants in 37
15
fa:rm fields and station trials.
The samples were desiccated over
16
CaCl, for serological studies and to provide sources of reference
17
isolates.
These samples and experimental seedlots from virus-
18
inoculated plants were subsequently shipped and/or brought to the
19
Virology Laboratory, Botany and Plant Pathology,
Oregon State
20
University, Corvallis, for investigation.
Plant Disease
Ndiaye,, et a1
Page 4
5
Se:rology.
Field samples from the five areas in Senegal were
tested by
either
double
antibody
sandwich
enzyme-linked
immunosorbent assay (DAS-ELISA) (5, 6) or by direct-antigen-coating
4
(DAC) E:LISA (8) for the possible presence of seven seed-borne
5
viruses
(Table 1):
blackeye cowpea mosaic (BlCMV) and cowpea
6
aphid-blorne mosaic (CABMV) potyviruses, cowpea mosaic (CPMV) and
7
cowpea severe mosaic (CSMV) comoviruses, cowpea mottle carmovirus
8
(CPMoV) , cucumber mosaic cucumovirus (cm I and southern bean
9
mosaic sobemovirus (SBMV).
The samples were also tested by DAC-
:10
ELISA against either potyvirus-selective monoclonal antibody (MAb)
11
Agdia PTY (11) or anti-BCMV MAb II-197 (21).
:12
Antisera to BlCMV and SBMV were kindly provided by Dr. Cedric
1 3
Kuhn; antisera to CPMV and CSMV were kindly provided by Dr. 0. W.
14
Barnett; and MAb II-197 was kindly provided by Dr. G. 1. Mink.
The
15
other antisera were produced by the Virology Laboratory, USDA ARS,
16
Dept of Botany & Plant Pathology, Oregon State University.
17
Virus isolates derived from infected seeds of four advanced
18
Senegal cowpea lines (Table 2, except that no seed-transmission was
19
observed in cv. IS86-283-15) were also tested by DAS-ELISA against
.2 0
immunogammaglobulin G (IgG) to BlCMV, CABMV, pea seed-borne mosaic
21
potyvirus (PSbMV) and CMV (as possible contaminant), and by DAC-
22
ELISA against antisera of the following potyviruses: BlCMV, CABMV,
23
clover yellow vein virus (CYW), peanut mottle virus (PeMV), peanut
Plant Disease
Ndiaye, et a1
Page 5
6
1
stripe virus (PStV), PSbMV, white lupin mosaic virus (WLMV) and
2
also against PTY MAbs.
Isolaies reactive in DAC-ELISA only to the
3
monoclo:nal antibodies were tested a second time by DAC-ELISA
4
for- possible contamination with CPMoV,
CPMV, CSMV and SBMV.
5
In the concluding phase of this study, antisera to unknown
6
potyvirus
isolate V17-14 were produced in two Young laying
7
chickens.
A series of five breast-intramuscular injections of 150
a
to 200 ug of purified potyvirus were made at weekly or bi-weekly
9
intervals.
Eggs were saved during a period of 10 weeks.
IgG was
10
extracted from the yolks of selected egg clutches, by the- methods
.ll
of Jensenius et a1 (10).
Four wk after the final injection, the
1 2
chickens were anesthetized and exsanguinated.
IgG derived from the
1 3
blood was compared with yolk-derived IgG, and blood-derived IgG was
14
chosen for comparisons of serological affinities among selected
15
BlCMV and CABMV and isolate V17-14, by DAS-ELISA (Table 4).
16
Absorbante values were reco:rded by a Bioteck Mode1 EL-309
17
ELISA reader, typically 90 min after addition of enzyme substrate,
18
p-nitrophenyl phosphate.
Tested antigens were buffered extracts
19
from fresh or desiccated tissues of virus-infected plants.
20
Electron microscopy.
Potyvirus-like virions were visualized
21
in leaf-dip extracts and partially purified virus preparations, by
22
means of a Philips EM 12 electron microscope.
For viewing, the
23
preparations were adsorbed to carbon-coated copper grids
and
Plant Dlisease
Ndiaye, et a1
Page 6
7
1
negatively stained with 2% ammonium molybdate (pH 7.0).
For
2
estimations of virion sizes, the scope was calibrated using both
3
interna1 and external magnification standards.
4
Disease reactions, seed transmission, and host range tests.
5
Seedling plants of five advanced cowpea lines/cultivars (Table ,2)
6
were dusted with silicon carbide powder and mechanically inoculated
7
with four Senegal viral field isolates, for tria1 reproduction jof
8
the previously observed disease symptoms.
Viral isolates V-l a:nd
9
V-2 were collected from naturally infected cowpea plants in Kolda,
3.0
and isolates V-17 and V-54-were taken from comparable plants in
1.1
Diourbe:L.
Inoculated plants were maintained under both field and
12
screenhouse conditions near Bambey, and were inoculated a seco,nd
13
time to increase the potential for virus transmission.
Insecticide
14
was applied as needed to control potential insect vectors.
Disease
15
incidence in plots (% symptomatic: plants) was recorded biweekly
16
from 7 to 45 days after inoculation.
17
Seeds were harvested from plants in these Isolate x Genotype
la
treatments and tested for seed-borne virus by growing out seedlings
19
in insect-free glasshouses, at Oregon State University.
Seedling
2 0
infection was examined first by visual inspection and then by
21
ELISA.
Two-wk-old symptomatic and selected viral-symptomatic
22
seedlings were assayed for seed-borne potyviruses by DAC-ELISA with
23
potyvirus-selective monoclonal antibodies Agdia PTY and 11-197.
Plant Disease
Ndiaye, et a1
Page 7
8
Potyviral isolates derived from individual infected seedlings
of cv 58-57 or Mougne (i.e., screenhouse-grown, inoculated mother
plants) were assigned distinct sub-numbers (Tables 3, 5, 6) and
thereafter referred to as PTY+ (i.e.,
detectable by monoclonal
antibody PTY).
Stock cultures of such isolates were preserved in
desiccated infected tissues at - :30 C and maintained in infected
seeds of: selected cowpea cultivars.
A small set of selected plant
species/genotypes (Table 5) and a strategic set of cowpea genotypes
9
(3, 4)(Table 6) were tested for susceptibility to five selected
10.
seed-borne PTY+ potyviral isolates.
Eight to ten plants of each
3.1
species/genotype
were
inoculated under glasshouse Conditio:ns
12
(temperature, 28-30 C; 14 hr photoperiod; and solar irradia:nt
3.3
equivalence of 87 to 121 kJm" day-'.
Symptomless inoculated plants
14
were assayed for asymptomatic infection, using Agdia PTY MAb 'by
15
DAC-ELISA, 5 wk after inoculation.
16
Aplaid transmission. Aphid-transmissibility of seed-borne PTY+
8-7
isolates Vl-1 and V17-14 was tested using an A@is craccivora
18
colony reared on healthy cowpea plants (Table 7).
Plant to plant
19
transmission was carried out as follows:
after a 2-hr fasting
20
period, groups of 4th or 5th instar apterae were deposited on
21
detached virus-infected cowpea leaves for 3 to 4 min acquisition
22
periods.
Apterae found in feeding position were then carefully
23
transferred with a fine camel-hair brush to healthy plants of
Plant Disease
Ndiaye, et a1
Page 8
9
Senegal cowpea cv 58-57.
For each virus isolate, 26 to 35 test
plants were inoculated using three aphids per test plant.
The
aphids were allowed to feed overnight on test plants before removal
4
by insecticide.
Inoculated plants were observed for symptom
developiment for 4 wk after aphid inoculations.
Symptomless
plants were assayed by DAC-ELISA using PTY MAb.
Each transmission
test was repeated.
8
RESULTS
9
Field survey and virus detection.
Of 66 samples collected
10
from five cowpea growing areas of Senegal, 36 reacted positively
11
with one or more of the seven test antisera or with MAb (Table 1).
12
Neither BlCMV, CMV, nor CPMV was detected among the Senegal test
13
samples.
Thirty-four of 66 (52%) samples contained CABMV.
One
:14
sample from Diourbel contained both CSMV and CPMoV.
SBMV was
15
detected in only one sample from Louga in mixture with CPMoV.
16
Twenty-one samples (32%) reacted with potyvirus MAb 11-197, blut
17
reacted with none of the seven polyclonal antibodies.
Based on
18
these unique reactions and supplementary serology, isolates W-1
19
and V17-14 were concluded to be an unknown potyviruses (i-e.,
20
designated PTY+).
This unknown potyvirus and CABMV were the
21
predominant cowpea viruses at a11 locations surveyed in Senegal.
Plant Disease
Ndiaye, et a1
Page 9
10
Inoculations and seed transmission.
The disease incidence in
meohanically virus-inoculated plants (screenhouse and field) of
five Senegal cowpea genotypes ranged from 8% to 100% (Table 2).
Cowpea line 1886-283-15 was partially resistant or tolerant to two
of four viral field isolates, Vl and V2.
Results from screenhouse
6
and field inoculations were essentially the same.
7
Seed transmission rates of the four potyvirus isolates in five
8
Sertegal cowpea genotypes varied from 0 to 30 % (Table 2).
Isolate
9
V17 was seed-transmitted in cv 58-57 at a rate of 30%; however,
10
larger :numbers of seeds are required to assess real differences in
11
seed-transmission rates among field isolates.
Senegalcv 58-57 was
12
most seed-transmission prone, whereas no seed transmission of PTY+
13
occurred in line IS86-283-15 N.
14
Serological relationships.
Seed-borne isolates of potyvirus
15
PTY+ were tested by DAS-ELISA and found to be serologically
16
unrelated to potyviruses BlCMV, CABMV or PSbMV, and also to be free
17
of ELISA detectable CMV (Table 3).
The same isolates reacted to
18
var-ying degrees in DAC-ELISA with antisera to five selected
19
potyviruses (BlCMV, CABMV, PeMV, PMV, PSbMV and PStV), but not with
20
antisera to CYVV,
or WLMV, as would be expected of this less
21
discriminating version of ELISA.
The PTY+ isolates were verified
22
to be free of ELISA-detectable CPMoV, CSMV, PStV, or SBMV.
Al1
23
PTY+ isolates reacted with both MAbs Agdia PTY and 11-197; however,
Plant Disease
Ndiaye, et a1
Page 10
II .,
:
. ^___ .._____-. -.. I.
-_
---
11
the reactions of isolate Vl-1 were consistently weaker with either
MAb.
IgG to PTY+ isolate V17-14 reacted indistinguishably to a11
sister isolates of PTY+.
However, this IgG reacted neither to 10
selected BlCMV isolates nor to 9 of 11 selected CABMV isolates
(Table 4).
Reactions by CABMV isolates RN-27C and RN-28C were
unexpected,
since they were previously considered typical, pure
8
CABMV isolates,
We did not determine whether the results indicated
9
the sha:ring of coat protein epitope between RN isolates and PTY+
10
Vl7-14 or contamination of RN isolates with PTY+.
Both RN isolates
11
had originated in cowpea seeds obtained from Botswana (3, 4).
12
Electron microscopy.
Plants infected with PTY+ isolates
13
contained flexuous rod-shaped particles, visualized by electron
14
microscopy in leaf dip or partially purified preparations.
The
15
modal length of >lOO particles was approximately 725 nm, thus
16
fitting within the recognized 710 to 900 nm size range of potyvirus
17
particles,
18
Preliminary host-range tests.
Few differences were found in
19
the host range/reactions of five seed-borne potyvirus isolates
20
(Table 5),
including minor variations in
symptoms
induced in
21
Senegal cowpea genotypes and Chenopodium amaranticolor Coste &
22
Reyn. and susceptibility to asymptomatic infection in Phaseodus
23
vulgaris L.
(bean cv Topcrop).
Generally,
the host ranges for
Plant Disease
Ndiaye, et a1
Page 11.
12
1
these isolates were more narrow than those for typical isolates of
2
cAJ3MV (3, 4).
3
Screening of cowpea cultivars for resistance.
The reactions
4
of cowpea lines/cultivars to mechanical inoculation with five seed-
5
borne PTY+ isolates were determined (Table 6).
Five International
6
Institute of Tropical Agriculture (IITA) TW lines, TW-401, TVU-
7
408P2,
TW-1000, TW-1016-1, and TW-1582 and one U.S.A. cv White
8
Acre-BVR were immune to a11 isolates (i-e., asymptomatic and free
9
of ELISA MAb-detectable virus.
Some genotypes were susceptible to
10
a11 PTY+ isolates.
Other genotypes were susceptible to specific
11
isolates, e.g., cv Serido was susceptible only to PTY+ V17-14; cv
12
TVlJ-410 was susceptible only to PTY+ V54-23, whereas TW 984 was
13
resistant only to this isolate.
No attempt was made to classify
14
PTY+ plathotypes
using these
cowpea genotypes;
however,
the
15
genotypes provided evidence that the isolates were pathogenically
16
diverse.
17
Aplhid transmission.
Seed-borne PTY+ isolates Vl-1 and V17-14
18
were
both
efficiently
transmitted
nonpersistently by
Aphis
19
craccivura.
In replicated triais,,
isolate Vl-l was transmitted to
20
>60% (42 of 61) of the plants inoculated by 3 apterae/plant and, in
21
parallel tests, isolate V17-14 was transmitted to 59 of 59 plants
22
inoculated.
We believe that A. craccivora is a probable vector of
23
ail seed-borne potyviruses in Senegal and that indigenous biotypes
Plant Disease
Ndiaye, et a1
Page 12
13
1
cari transmit isolates of potyvirus PTY+ at rates comparable to
2
these experimental results.
3
DISCUSSION
4
Cowpea
viruses
are increasingly
important in
a11 cowpea
growing areas of Senegal.
The survey reported herein was prompted
because new pathogen/pest-resistant breeding lines had been damaged
by viral diseases.
Moreover, seed-borne viruses were designated
priority pathogens in these studies, since they have historically
9
inflict'ed heavy losses through unknowing establishment of seed-
10
borne field inoculum, followed by secondary spread by insect-vector
11
species (8).
12
The present study of 66 strategic cowpea samples with virus-
:13
1ik.e symptoms indicated the presence in Senegal of four recognized
:14
seed-borne viruses, CABMV, CSMV, CPMoV, and SBMV, and an apparently
15
new potyvirus herein designated PTY+.
SBMV had already been
16
reported from the Casamance regiom of Senegal (7).
Hampton et a1
1 7
(8), had detected CSMV in cowpea germplasm accessions from Senegal.
18
CPMoV was previously reported only from Nigeria (1, 18), until
19
recently when it was reported in Pakistan-grown cowpeas (2). CPMoV
20
was also detected in cowpea
sa'mples collected from screening
Plant Disease
Ndiaye, et a1
Page 13
----.. -.
--
.
-.
14
nurseries in Riverside, California, U.S.A (M. Bashir, unpublished
results).
The seed-borne nature of CPMoV (1, 18) and these recent
detections suggest that the virus is now spreading through seeds to
other parts of the world.
However, CABMV and potyvirus PTY+ were
the prevalent viruses, occurring in 83% of the 66 samples and
accounting for 55 of the 57 samples in which viruses were ELISA-
detected.
Based on prior investigations (8,
and unpublished
8
results), CMV and CPMV were expected to occur in Senegal-grown
9
cowpeas,
but neither was detected.
3.0
Multiple-virus infections tendto be common among samples from
11
field-grown cowpeas, world-wide.
Such mixed infections are known
12
to modify and complicate symptoms, essentially precluding field
1.3
diagnosis (9, 12).
In these studies, however, mixtures of seed-
14
borne viruses were found in only two of the 66 cowpea tissue
7.5
samples, CSMV + CPMoV and SBMV + CPMoV.
3.6
The five seed-borne PTY+ potyvirus isolates examined by DAS-
3.7
ELISA did not react with IgGs to CABMV and BlCMV (Table 3), a:nd
18
most CA:BMV and BlCMV isolates were non-reactive to IgG to PTY+
19
isolate V17-14 (Table 4).
Despite clear serological distinctions
;! 0
among these three viruses, the interactions of PTY+ isolates with
21
cowpea Igenotypes resembled CABMV, lacking the ability to infect
22
IITA accessions TVU-401 and TVU-1582, previously proven resistant
23
to a11 tested CABMV isolates (3, 4).
It is therefore conceivable
Plant Disease
Ndiaye, et a1
Page 14
c
15
1
that PTY+ is a distinct serotype and pathotype of CABMV and that
2
TVIJ-401 and TVU-1582 contain multiple genes/alleles against a11
3
tested CABMV variants.
4
While various controlmeasures may impede cowpea viraldisease
5
development, including control of insect vectors, removing diseased
6
plants from seed fields, and production of virus-free seed, we
7
believe the development of resistant cultivars
is the most
8
practical and economical control measure for such diseases. In
9
this situdy,
we identified six cowpea genotypes as
sources of
10
resistance to a11 isolates of PTYi-.
These genotypes have now been
11
incorporated in the extant Senegal cowpea breeding program for
12
development of
improved disease/pest-resistant
cultivars
for
13
Senegal cowpea production areas.
Plant Disease
Ndiaye, et a1
Page 15,
_-.-.
.-.
-7-
._
16
LITERA- CITED
1.
Allen, D.G., Thottappilly, G. and Rossel, H.W. 1982.
Cowpea
mottle virus: field resistance and seedtransmission in virus-
to:Lerant cowpea. Ann. Appl. Biol. 100:331-336.
2.
Bashir, M., and Hampton, R.O. X991.
Natural occurrence of five
cowpea
viruses in
Pakistan.
Phytopathology
81:1166-1167
(Abstr.) .
3.
Bashir, M., and Hampton, R. O.,
1991.
Blackeye cowpea mosaic
and
cowpea
aphidborne
mosaic
potyviruses:
Biological
comparisons
and serological
distinctions.
Phytopathology
81::L166 (Abstr.).
4.
Bashir, M.,
and
Hampton, R. 0.
1992.
Biological
characterization of' pathotypes of blackeye cowpea mosaic and
cowpea aphidborne mosaic potyviruses.
Phytopathology 82:1103
(Abstr.) .
5.
Clark, M.F. and Adams, A.N. 1977.
Characteristics of the
microplate method of enzyme-linked immunosorbent assay for the
detection of plant viruses. J., Gen. Virology. 34:475-483.
6.
Converse, R.H. and Martin, R.R.
1990.
ELISA methods for plant
viruses. In *‘
Serological
methods
for
detection
and
identification of viral and bacterial plant pathogens, A
laboratory manual. Hampton,R.O., Ball,S., and Boer, S. (Eds).
APS Press U.S.A.
389 pp.
7.
Gaikwad, D-G.
and Thottappilly, G.
1987.
Occurrence of
southern bean mosaic Virus on cowpea in
Senegal.
J.
Phytopathology. 120: 81-84.
8.
Hampton, R.O., Albrechtsen, S.E. and Mathur, S.B.
1992.
Seed
health (Viruses) of Viqna unsuiculata cultivars/selections from
developing countries.
Seed Science & Technol. 20:23-38.
9.
Har:rison, A.N. and Gudauskas, R.T.
1968.
Effects of some
viruses on growth and seed production of two cowpea cultivars.
Pla:nt Dis. Reptr. 52:509-511.
Plant Disease
Ndiaye, et a1
Page 16
c
17
1 0 .
Jensenius, J.C., Anderson, I., Hau, J., Crone, M., and Koch,
CI
1981.
Conveniently packaged antibodies.
Methods for
purification of yolk IgG.
Jour. of Immunol. Meth. 46:63-68.
1 1 .
Jordan, R.
1992.
Potyviruses,
monoclonal antibodies, and
antigenic sites.
Arch. Virol. (Suppl. 5):81-95.
12. Kuhn, C.W., Brantley, B.B. and G. Sowell, Jr.
1966.
Southern
pea viruses:
identification,
symptomatology and sources of
resistance.
Georgia Agric.
:Expt.
Stn.
Bull.
157,
Athens,
Georgia. 22 pp.
13. Lana, A.F.
and Adegbola,
M.O.K.
1977.
Important virus
diseases in West African crops. Rev. Plant Pathology 56:849-
865,
14. Lamptey, V-R. and Hamilton, B.L.
1974.
A new cowpea strain of
southern bean mosaic virus from Ghana.
Phytopathology 64:1100-
1104.
15. Mali, V.R. and Thottappilly, G.
1986.
Viruses on cowpea in
the tropics.
Rev. Tropical Plant Pathology 34: 421-522.
16. Ndiaye, M., Bashir, M., Keller, K. K., and Hampton, R. 0.
1992.
Identification, distribution and seed transmission of
cowpea viruses in Senegal.
Phytopathology 82:1103 (Abstr.).
17. Raheja, A.K. and Leleji, 0.1.
1974.
An aphid-borne mosaic
disease of irrigated cowpeas in northern Nigeria.
Plant
Disease 58: 1080-1084.
18. Shoyinka, S.A., Bozarth, R.F., Reese, J. and Rossel, H.W.
1978 a
Cowpeas
mottle
virus: A
seed-borne
virus
with
distinctive
properties
infecting
cowpea in
Nigeria.
Phytopathology 68:693-699.
19.
Thiaw, S., Hall, A.E.,
and Parker, D.R.
1993.
Varietal
intercropping andthe yields and stability of cowpea production
in semiarid Senegal.
Field Crops Research (in press).
20. Thottappilly, G. and Rossel, H.W.
1985.
World occurrence and
distribution of virus diseases.
In Cowpea Research, production
and Utilization.
Singh, S.R., Rachie, K.O. (Eds.). John Wiley
& Sons, Chichester, Great Britain, London.
Plant Disease
Ndiaye, et a1
Page 17
._----”- .--- --_“-
f
18
21. Wang, W., Mink, G. I., Silbernagel, M. J., and Davis, W. 12.
1984.
Production
of hybridoma
lines
secreting
specific
antibodies to bean common mosaic virus
(BCW
strains,
Phytopathology 74:1142. (Abstr.).
22. WillLiams, R. J.
1975.
Diseases of cowpea [ Vigna unguiculaiza
(L.) Walp. ] in Nigeria. PANS 21~253-257.
Plant Disease
Ndiaye, et a1
Page 18
.--_
--
-- . . ..-
Table 1.
Viruses detected by DAC-ELISA in field samples of cowpea collected in
five districts of Senegal (West Africa)
No. of samples reacting positively with antiviral
antisera/potyvirus MAb.
District
No.
-------------------------------------------------------------
surveyed/
samples
BlCMV CABMV CMV
CPMoV
CPMV
CSMY
SBMV
MAB
sampled
collected
II-197
Diourbel
28
- 1
10
-
1
1
11 2
Kolda
4
1
-
2
Louga
20
10
-
1
2
1
7
Tambacounda 10
9
-
1
Thies
4
4
-
Total
66
34
-
2
1
1
21
1
-
I
indicates virus not detected by ELISA.
2
Samples reacting to monoclonal antibody II-197 (21) contained no virus
detectable by other anti-viral polyclonal antisera.
Plant Disease
Ndiaye, et a1
Page 19
.
.J
<,;yr:.r
-.-i.,,
--------------------_I___________)c_cc__-------------~-~--~~--~--------------~--
Table 2.
Disease incidence' and seed transmission2 associated with Senegal
potyvirus PTY+ field isolates
Disease
No. of seeds
Seed
Virus
Cultivars/
incidence
germinated/
transmission
isolate lines
(%)
planted
Incidence %
v 1
Baye Ngagne
100
37/50
1137
3
IS86-275N
61
41/50
0/41
0
IS86-283-15
8
29/50
0/29
0
Mougne
97
46/50
2146
4
58-57
100
46150
6146
13
v 2
Baye Ngagne
100
46/100
0/46
0
IS86-275N
47
37/100
0137
0
1686-283-15
18
44/100
0144
0
Mougne
100
921100
1192
1
58-57
55
831100
7183
8
v 17
Baye Ngagne
81
035/50
0/35
0
IS86-275N
50
40/50
2/40
5
1886-283-15
52
23/50
0/23
0
Mougne
97
47150
0147
0
58-57
93
43/50
13/43
30
v 54
Baye Ngagne
86
27/50
0127
0
IS86-275N
88
40/50
2/40
5
1586-283-15
82
28150
0/28
0
Mougne
97
49/50
0/49
0
58-57
89
44/50
1/44
2
' Experiments conducted in field and screenhouse plots, Bambey, Senegal.
2
Seeds taken from potyvirus inoculated plants, Bambey screenhouse plots.
Experiments
conducted in greenhouses, Corvallis, OR, U.S.A.
0
Plant Disease
Ndiaye, et a1
Page 20
Table 3.
CCRilFiXiSQnS ^f eight B2CîrJniZed F@tyV iruses with five seed-borne Senegal
cowpea potyvirus isolates, by DAS- and DAC-ELISA.
The Senegal isolates were also
tested for the possible presence of four seed-borne non-potyviruses
A 4O5 values '
Seed-borne isolates of Senegal potyvirus, PTY+
Homolo-
Healty-
gous
plant
Antiserum
Vl-1
V17-2
v17-14
v54-3
V54-23
virus 2
extract
DAS-ELISA
BlCMV 3
0.00
0.01
0.01
0.00
0.01
0.95
0.01
CABMV
0.01
0.01
0.01
0.01
0.03
1.15
0.01
PSbMV
0.00
0.00
0.00
0.00
0.00
1.87
0.00
CM-V
0.01
0.00
0.01
0.00
0.00
1.27
0.02
DAC-ELISA
BlCMV
0.26
1.20
0.36
0.37
0.21
2.81
0.02
CABMV
1.68
1.65
1.51
1.30
1.41
2.48
0.02
CYW
0.11
0.10
0.08
0.05
0.08
>3.00
0.01
PeMoV
1.63
1.38
2.03
1.27
1.58
>3.00
0.03
Pstv
1.90
1.50
1.75
1.10
1.92
>3.00
0.02
PM-V
1.86
0.54
0.23
0.78
0.51
>3.00
0.02
PSbMV
1.65
1.58
1.51
1.86
2.11
>3.00
0.01
WLMV
0.01
0.02
0.01
0.02
0.04
>3.00
0.01
CPMoV
0.03
0.02
0.02
0.02
0.00
>3,00
0.03
CSMV
0.16
0.10
0.11
0.15
0.13
>3.00
0.12
SBMV
0.00
0.00
0.00
0.00
0.02
>3.00
0.01
II-197
0.31
0.77
1.40
0.57
0.70
0.58 4
0.00
(BCMV MAb)
Agdia
0.22
NT 5
0.70
0.78
0.86
0.77 6
0.00
(PTY MAb)
Plant Disease
Ndiaye, et a1
Page 21
Table 3; cent.
1
Ado values recorded after 90 min incubation with substrate, p-nitrophenyl
phosphate.
2
Virus homologous to each antiserum, e.g., the viral homologue of BlCMV
antiserum is blackeye cowpea mosaic potyvirus.
3
Antisera to potyviruses are:
BlCMV, blackeye cowpea mosaic virus; CABMV,
cowpea aphid-borne mosaic virus; CYW, clover yellow vein virus; PMV, pea
mosaic virus, PeMoV, peanut mottle virus; PSbMV, pea seedborne mosaic virus;
PStV, peanut stripe virus; and WLMV, white lupin mosaic virus.
Antisera
to other viruses seed-borne in cowpea are:
CMV, cucumber mosaic virus; CSMV,
cowpea severe mosaic virus; SBMV, southern bean mosaic virus; and CPMoV, cowpea
mottle virus.
The Agdia monoclonal antibody (11) reacts to >90% of a11 tested
potyviruses; monoclonal antibody II-197 (21), produced against bean common
mosaic virus, reacts to its homologue and several other potyviruses.
4
BlCMV isolate RF-26B was selected as a positive control for MAb 11-197.
5
NT = Not tested.
6 CABMV isolate 9-7C was selected as a positive control for the Agdia PTY MAb.
Plant Diesease
Ndiaye, et a1
Page 22
23
Table 4.
DAS-ELISA tests of selected BlCMV and CABMV
isolates against chicken anti-V17-14 immuno-
gammaglobulin G
-7
Virus
isolate
A= values
B:LCMV-Ga '
0.003
PI-3B
0.005
RF-4B
0.012
PU-7B
0.004
PIJ-8B
0.003
PI-22B
0.007
PIC-23B
0.002
PI-25B
0.003
RI?-26B
0.002
RF-27B
0.007
RN-7c ’
RN-1oc
2
RN-27C
RN-28C
RN-34c
RN-35c
RN-36C
RN-37c
PI-39c
PI-4oc
PI-44C6
V:L7-14 (Homologue)
Healthy-plant
extract
' Isolates BlCMV-Ga (Georgia) and RN-7C (Botswana) were
included as type isolates of BlCMV and CABMV,
respectively.
2
Isolates RN-27C and RN--28C previously had reacted in
DAS-ELISA only to CABAN antiserum produced against
CABMV isolate RN-7C.
Plant Disease
Ncliaye, et a1
Pa&ge 23
-. _._------
Table 5.
Reactions by selected plant species to inoculations with five seed-borne
isolates of Senegal potyvirus PTY+
Disease reactions to PTY+ isolates
Host species
VI-1
W-7-2
v17-14 v54-3
V54-23
Lequminous hosts:
Lupinus albus cv Astra
- 1
Medicago sativa cv DuPuits
Trifolium pratense cv Kenland
Phaseolus vulgaris cv Monroe
Phaseolus vulgaris cv Top Crop
Vicia faba CV Hertz Freya
VN
VN
Non-lequminous hosts:
Chenopodium amaranticolor,
LLn
LLn.VN LLn
LLn,VN
Corvallis strain
Nicotiana benthamiana, ATCC
SM
S M
SM
S M
SM
Gomphrena globosa, A.F. Ross Strain
Phlox drumondii cv Ta11 Mixed Color
Lycopersicon esculentum cv Marglobe
Petunia hybrida cv King Henry
Antirrhinum majus cv Mixed Colors
1
Symbols are: - , no symptoms and no ELISA-detectable virus; LI, latent
(asymptomatic), ELISA-detectedinfection; LLn, necrotic locallesions; VN, vein
necrosis; SM, systemic mosaic.
2
Plant Disease
bidiaye, et a1
Page 24
25
Table 6.
Responses of selected cowpea genotypes to green-
house mechanical inoculations with five seed-
borne isolates of Senegal potyvirus PTY+
-
Potyvirus isolates
C o w p e a
genotypes
Vl-1
V17-2
v17-14
v54-3
V54-23
TVU 109P2 '
+ +
T V U 1 9 6
++
Tvu 347
+ +
++
Tvu 354
+ +
++
Tvu 401
TVU 408P2
Tw 410
TVU 984
+ +
++
TV-U 1000
TVU 1016-L
TVU 1582
TVU 2657
.
+ +
++
Tvu 3433
+ +
++
IT 81D 1137
+ +
++
IT 86 27N
+ +
++
P:I 25122
+ +
++
Bambey 21
++
Serido
Wlh Acre BVR
Cal Bl # 5
0
++
Snapper
0
++
Blue Goose
0
Corona
0
Mlopod
0
Tex Cr # 8
0
Tlex Cr # 40
0
UCR 524B
0
Mis Purple
0
Mis Silver
0
Magnolia
0
K.n Pur Hull
0
Worthmore
0
Bettergreen
0
UCR 7964
0
1
TW genotypes kindly provided by I.I.T.A., Ibadan, Nigeria.
‘2
Symbols are: - , no symptoms and no virus detectable by ELISA,
.1-e., immune; + , mild systemic symptoms; ++, moderate systemic
symptoms; LI, latent (asymptomatic),
ELISA-detected infection;
i-e.,
tolerant to infection; 0, not tested.
Plant Disease
Ndiaye, et a1
Page 25
COWPEA WIRUSES IN SENEGAL, WEST AFRICA:
IDENTITIES, DISTRIBUTION,
SEED-TRANSMISSION, AND SOURCES OF GENETIC RESISTANCE
M. Ndiaye, M. Bashir, X.E. Keller and R.O. Hampton
First author: Institut Senegalais de Recherches Agricoles / CNRA
de Bambey B.P. 53, Bambey, Senegal.
Second author: formerly
Department of Botany and Plant Pathology, Oregon State
University; nowNationa1 Agricultural Research Center, Islamabad,
Pakistan.
Third and fourth authors: U.S. Department of
Agriculture, Agricultural Research Service, Department of Botany
and Plant Pathology, Oregon State University, Corvallis OR
97331-2902.
Contribution of the USDA Agricultural Research Service in
cooperation with the Department of Botany and Plant Pathology,
Agricultural Experiment Station, Oregon State University,
Corvallis.
Technical paper 10177, Oregon Agricultural Experiment
Station.
This research was partially supported by the Bean/Cowpea Collabora-
tive Research Support Program of the United States Agency for
International Development, Grant No. DAN-131D-SS-6008-00.
Mention of a trademark or proprietary product does not constitute
a guarantee or warranty of the product by the U.S. Department of
Agriculture and does not imply its approval to the exclusion of
other products that also may be suitable.
ABSTRACT
Ndiaye, M., Bashir, M., Keller, K., and Hampton, R. 0.
1993 ?
Cowpea viruses in Senegal, West Africa:
Identities, distribution,
seed-transmission,
and sources of genetic resistance.
1
Viral diseases of cowpea in Senegal were surveyed during the
2
rainy seasons of 1990 and 1991.
Sixty-six viral-symptomatic plant
3
samples from five cowpea production areas were assayed for seven
4
viruses by DAC- or DAS-ELISA.
The following four recognized
5
viruses were detected, a11 of which are seed-transmissible in Vicma
6
unauiculata:
cowpea aphid-borne mosaic potyvirus (CABMV) (34/66),
7
cowpea
mottle carmovirus
(CPMoV) (2/66), cowpea severe mosaic
:
. .
)
i’
*
!p
?? ? ?
? ? ? ? ? ? ?
?
?
?
?
?
?
??
? ? ? ? ? ?
c
comovirus
(CSMV)(1/66), and
sou,thern bean mosaic sobemovirus
(SBMV)(:L/66).
In addition to these four viruses, variants of an
unknown potyvirus were detected in 21 of the 66 samples by use of
potyvirus-selective monoclonal antibodies (Agdia PTY and BCMV II-
5
197).
These potyvirus variants occurred principally in new
6
improved CABMV-resistant cowpea genotypes,
and their combin'ed
7
incidence was exceeded in plant samples only by CABMV.
Isolates of
8
the unknown potyvirus were seed-borne in Senegal cowpea lines and
9
were efficiently transmitted non-persistently by the cowpea aphid,
10
Aphis craccivora.
Selected seed-borne isolates of this potyuirus
11
were distinguishable principally by differentially resistantcowpea
x2
genotypes and by either weak (isolate Vl-1) or strong (isolate V17-
13
14)
reactions to
potyvirus-selective
monoclonal
antibodies.
14
Thirty-five
selected cowpea genotypes were tested as possible
15
sources of resistance to the unknown potyviruses.
Of these, six
16
(TVU-401, TW-408P2, TVU-1000, TW-1016-1, TW-1582, and White
17
Acre-BVR) were resistant to a11 isolates of the potyvirus.
These
X8
genotypes have been
included in the existing Senegal cowpea
19
breeding program for development of virus-resistant cultivars.
20
--
-
21
Cowpea [Vigna unguiculata (L.) Walp.] is second in importance
22
only to groundnut, among Senegal legume crops.
Senegal cowpeas are
23
annually grown on some 63,000 hectares, with an annual production
Plant Disease
Ndiaye, et a1
Page 2
3
1
of 18,C~OO,OOO kg.
Doubling the cowpea yield there should be
2
readily achievable, since the average yield of 280 kg/ha represents
3
only 14 to 28% of the 1,000 to 2,000 kg/ha yields in experimental
4
fields, in Senegal (19).
Several factors contribute to low yields
5
of cowpea in Senegal, and viral diseases are considered a major
'6
limitation.
7
Among cowpea viruses reported in West Africa (13, 15, 17, 1.9,
8
22) I cowpea yellow mosaic comovirus (CPYMV) and cowpea aphid-borne
9
mosaic
potyvirus
(CABMV)
are
considered
economically
most
10
important.
Other viruses known to occur in West Africa include
11
cowpea :mottle carmovirus (CPMoV) (1, 18) and southern bean mosa,ic
12
sobemovirus (SBMV) (7, 14).
Cowpea mild mottle carlavirus (CMMV),
13
cowpea
severe
mosaic
comovirus
(CSMV), and cucumber mosaic
14
cucumovirus (CMV) had been previously detected in seed lots from
15
Burkina Faso, Nigeria, Senegal, and Ghana, respectively (8).
16
Seed-borne viruses were considered a major constraint to
17
cowpea yield in Senegal farm fields (Gaikwad, D. G., unpublished
18
results).
Seed-borne viruses are especially destructive because
19
emerging plants are exposed early to seed-borne inoculum that is
20
acquired and progressively spread by insect vectors, particularly
21
by aphid and beetle species.
Two cowpea breeding lines, IS86-275N
22
(released in 1992 as cvMouride) and IS86-283-15N had been recently
23
developed to increase sustainable cowpea production in Senegal.
Plant Disease
Ndiaye, et a1
Page 3
.A.,
‘,.. , .; :,.
>..Oh
:
<i
_.---.~---.
--.
4
These lines were resistant to CABMV, bacterial blight (Xanthomonas
campestzis p v
vignicola) ,
storage
weevil
(Callosobruchus
maculatxs),
striga (Striga gesnerioides), and drought (19).
However, viral diseases in field trials of these lines in 1989-90
suggested the incidence of unrecognized indigenous viruses or
perhaps undescribed pathotypes of CABMV.
The present study was
conducted to isolate and partially characterize these viruses and
to identify resistant cowpea genotypes for use in developing virus-
resistant cultivars (16).
10
Materials and IMethods
11
Field survey and collection of viral isolates.
During the
12
rainy seasons of 1990 and 1991 fields were surveyed for viral
13
diseases in the five cowpea production areas of Senegal.
A total
14
of 66 samples was collected from viral symptomatic plants in 37
15
fa:rm fields and station trials.
The samples were desiccated over
16
CaCl, for serological studies and to provide sources of reference
17
isolates.
These samples and experimental seedlots from virus-
18
inoculated plants were subsequently shipped and/or brought to the
19
Virology Laboratory, Botany and Plant Pathology,
Oregon State
20
University, Corvallis, for investigation.
Plant Disease
Ndiaye,, et a1
Page 4
5
Se:rology.
Field samples from the five areas in Senegal were
tested by
either
double
antibody
sandwich
enzyme-linked
immunosorbent assay (DAS-ELISA) (5, 6) or by direct-antigen-coating
4
(DAC) E:LISA (8) for the possible presence of seven seed-borne
5
viruses
(Table 1):
blackeye cowpea mosaic (BlCMV) and cowpea
6
aphid-blorne mosaic (CABMV) potyviruses, cowpea mosaic (CPMV) and
7
cowpea severe mosaic (CSMV) comoviruses, cowpea mottle carmovirus
8
(CPMoV) , cucumber mosaic cucumovirus (cm I and southern bean
9
mosaic sobemovirus (SBMV).
The samples were also tested by DAC-
:10
ELISA against either potyvirus-selective monoclonal antibody (MAb)
11
Agdia PTY (11) or anti-BCMV MAb II-197 (21).
:12
Antisera to BlCMV and SBMV were kindly provided by Dr. Cedric
1 3
Kuhn; antisera to CPMV and CSMV were kindly provided by Dr. 0. W.
14
Barnett; and MAb II-197 was kindly provided by Dr. G. 1. Mink.
The
15
other antisera were produced by the Virology Laboratory, USDA ARS,
16
Dept of Botany & Plant Pathology, Oregon State University.
17
Virus isolates derived from infected seeds of four advanced
18
Senegal cowpea lines (Table 2, except that no seed-transmission was
19
observed in cv. IS86-283-15) were also tested by DAS-ELISA against
.2 0
immunogammaglobulin G (IgG) to BlCMV, CABMV, pea seed-borne mosaic
21
potyvirus (PSbMV) and CMV (as possible contaminant), and by DAC-
22
ELISA against antisera of the following potyviruses: BlCMV, CABMV,
23
clover yellow vein virus (CYW), peanut mottle virus (PeMV), peanut
Plant Disease
Ndiaye, et a1
Page 5
6
1
stripe virus (PStV), PSbMV, white lupin mosaic virus (WLMV) and
2
also against PTY MAbs.
Isolaies reactive in DAC-ELISA only to the
3
monoclo:nal antibodies were tested a second time by DAC-ELISA
4
for- possible contamination with CPMoV,
CPMV, CSMV and SBMV.
5
In the concluding phase of this study, antisera to unknown
6
potyvirus
isolate V17-14 were produced in two Young laying
7
chickens.
A series of five breast-intramuscular injections of 150
a
to 200 ug of purified potyvirus were made at weekly or bi-weekly
9
intervals.
Eggs were saved during a period of 10 weeks.
IgG was
10
extracted from the yolks of selected egg clutches, by the- methods
.ll
of Jensenius et a1 (10).
Four wk after the final injection, the
1 2
chickens were anesthetized and exsanguinated.
IgG derived from the
1 3
blood was compared with yolk-derived IgG, and blood-derived IgG was
14
chosen for comparisons of serological affinities among selected
15
BlCMV and CABMV and isolate V17-14, by DAS-ELISA (Table 4).
16
Absorbante values were reco:rded by a Bioteck Mode1 EL-309
17
ELISA reader, typically 90 min after addition of enzyme substrate,
18
p-nitrophenyl phosphate.
Tested antigens were buffered extracts
19
from fresh or desiccated tissues of virus-infected plants.
20
Electron microscopy.
Potyvirus-like virions were visualized
21
in leaf-dip extracts and partially purified virus preparations, by
22
means of a Philips EM 12 electron microscope.
For viewing, the
23
preparations were adsorbed to carbon-coated copper grids
and
Plant Dlisease
Ndiaye, et a1
Page 6
7
1
negatively stained with 2% ammonium molybdate (pH 7.0).
For
2
estimations of virion sizes, the scope was calibrated using both
3
interna1 and external magnification standards.
4
Disease reactions, seed transmission, and host range tests.
5
Seedling plants of five advanced cowpea lines/cultivars (Table ,2)
6
were dusted with silicon carbide powder and mechanically inoculated
7
with four Senegal viral field isolates, for tria1 reproduction jof
8
the previously observed disease symptoms.
Viral isolates V-l a:nd
9
V-2 were collected from naturally infected cowpea plants in Kolda,
3.0
and isolates V-17 and V-54-were taken from comparable plants in
1.1
Diourbe:L.
Inoculated plants were maintained under both field and
12
screenhouse conditions near Bambey, and were inoculated a seco,nd
13
time to increase the potential for virus transmission.
Insecticide
14
was applied as needed to control potential insect vectors.
Disease
15
incidence in plots (% symptomatic: plants) was recorded biweekly
16
from 7 to 45 days after inoculation.
17
Seeds were harvested from plants in these Isolate x Genotype
la
treatments and tested for seed-borne virus by growing out seedlings
19
in insect-free glasshouses, at Oregon State University.
Seedling
2 0
infection was examined first by visual inspection and then by
21
ELISA.
Two-wk-old symptomatic and selected viral-symptomatic
22
seedlings were assayed for seed-borne potyviruses by DAC-ELISA with
23
potyvirus-selective monoclonal antibodies Agdia PTY and 11-197.
Plant Disease
Ndiaye, et a1
Page 7
8
Potyviral isolates derived from individual infected seedlings
of cv 58-57 or Mougne (i.e., screenhouse-grown, inoculated mother
plants) were assigned distinct sub-numbers (Tables 3, 5, 6) and
thereafter referred to as PTY+ (i.e.,
detectable by monoclonal
antibody PTY).
Stock cultures of such isolates were preserved in
desiccated infected tissues at - :30 C and maintained in infected
seeds of: selected cowpea cultivars.
A small set of selected plant
species/genotypes (Table 5) and a strategic set of cowpea genotypes
9
(3, 4)(Table 6) were tested for susceptibility to five selected
10.
seed-borne PTY+ potyviral isolates.
Eight to ten plants of each
3.1
species/genotype
were
inoculated under glasshouse Conditio:ns
12
(temperature, 28-30 C; 14 hr photoperiod; and solar irradia:nt
3.3
equivalence of 87 to 121 kJm" day-'.
Symptomless inoculated plants
14
were assayed for asymptomatic infection, using Agdia PTY MAb 'by
15
DAC-ELISA, 5 wk after inoculation.
16
Aplaid transmission. Aphid-transmissibility of seed-borne PTY+
8-7
isolates Vl-1 and V17-14 was tested using an A@is craccivora
18
colony reared on healthy cowpea plants (Table 7).
Plant to plant
19
transmission was carried out as follows:
after a 2-hr fasting
20
period, groups of 4th or 5th instar apterae were deposited on
21
detached virus-infected cowpea leaves for 3 to 4 min acquisition
22
periods.
Apterae found in feeding position were then carefully
23
transferred with a fine camel-hair brush to healthy plants of
Plant Disease
Ndiaye, et a1
Page 8
9
Senegal cowpea cv 58-57.
For each virus isolate, 26 to 35 test
plants were inoculated using three aphids per test plant.
The
aphids were allowed to feed overnight on test plants before removal
4
by insecticide.
Inoculated plants were observed for symptom
developiment for 4 wk after aphid inoculations.
Symptomless
plants were assayed by DAC-ELISA using PTY MAb.
Each transmission
test was repeated.
8
RESULTS
9
Field survey and virus detection.
Of 66 samples collected
10
from five cowpea growing areas of Senegal, 36 reacted positively
11
with one or more of the seven test antisera or with MAb (Table 1).
12
Neither BlCMV, CMV, nor CPMV was detected among the Senegal test
13
samples.
Thirty-four of 66 (52%) samples contained CABMV.
One
:14
sample from Diourbel contained both CSMV and CPMoV.
SBMV was
15
detected in only one sample from Louga in mixture with CPMoV.
16
Twenty-one samples (32%) reacted with potyvirus MAb 11-197, blut
17
reacted with none of the seven polyclonal antibodies.
Based on
18
these unique reactions and supplementary serology, isolates W-1
19
and V17-14 were concluded to be an unknown potyviruses (i-e.,
20
designated PTY+).
This unknown potyvirus and CABMV were the
21
predominant cowpea viruses at a11 locations surveyed in Senegal.
Plant Disease
Ndiaye, et a1
Page 9
10
Inoculations and seed transmission.
The disease incidence in
meohanically virus-inoculated plants (screenhouse and field) of
five Senegal cowpea genotypes ranged from 8% to 100% (Table 2).
Cowpea line 1886-283-15 was partially resistant or tolerant to two
of four viral field isolates, Vl and V2.
Results from screenhouse
6
and field inoculations were essentially the same.
7
Seed transmission rates of the four potyvirus isolates in five
8
Sertegal cowpea genotypes varied from 0 to 30 % (Table 2).
Isolate
9
V17 was seed-transmitted in cv 58-57 at a rate of 30%; however,
10
larger :numbers of seeds are required to assess real differences in
11
seed-transmission rates among field isolates.
Senegalcv 58-57 was
12
most seed-transmission prone, whereas no seed transmission of PTY+
13
occurred in line IS86-283-15 N.
14
Serological relationships.
Seed-borne isolates of potyvirus
15
PTY+ were tested by DAS-ELISA and found to be serologically
16
unrelated to potyviruses BlCMV, CABMV or PSbMV, and also to be free
17
of ELISA detectable CMV (Table 3).
The same isolates reacted to
18
var-ying degrees in DAC-ELISA with antisera to five selected
19
potyviruses (BlCMV, CABMV, PeMV, PMV, PSbMV and PStV), but not with
20
antisera to CYVV,
or WLMV, as would be expected of this less
21
discriminating version of ELISA.
The PTY+ isolates were verified
22
to be free of ELISA-detectable CPMoV, CSMV, PStV, or SBMV.
Al1
23
PTY+ isolates reacted with both MAbs Agdia PTY and 11-197; however,
Plant Disease
Ndiaye, et a1
Page 10
II .,
:
. ^___ .._____-. -.. I.
-_
---
11
the reactions of isolate Vl-1 were consistently weaker with either
MAb.
IgG to PTY+ isolate V17-14 reacted indistinguishably to a11
sister isolates of PTY+.
However, this IgG reacted neither to 10
selected BlCMV isolates nor to 9 of 11 selected CABMV isolates
(Table 4).
Reactions by CABMV isolates RN-27C and RN-28C were
unexpected,
since they were previously considered typical, pure
8
CABMV isolates,
We did not determine whether the results indicated
9
the sha:ring of coat protein epitope between RN isolates and PTY+
10
Vl7-14 or contamination of RN isolates with PTY+.
Both RN isolates
11
had originated in cowpea seeds obtained from Botswana (3, 4).
12
Electron microscopy.
Plants infected with PTY+ isolates
13
contained flexuous rod-shaped particles, visualized by electron
14
microscopy in leaf dip or partially purified preparations.
The
15
modal length of >lOO particles was approximately 725 nm, thus
16
fitting within the recognized 710 to 900 nm size range of potyvirus
17
particles,
18
Preliminary host-range tests.
Few differences were found in
19
the host range/reactions of five seed-borne potyvirus isolates
20
(Table 5),
including minor variations in
symptoms
induced in
21
Senegal cowpea genotypes and Chenopodium amaranticolor Coste &
22
Reyn. and susceptibility to asymptomatic infection in Phaseodus
23
vulgaris L.
(bean cv Topcrop).
Generally,
the host ranges for
Plant Disease
Ndiaye, et a1
Page 11.
12
1
these isolates were more narrow than those for typical isolates of
2
cAJ3MV (3, 4).
3
Screening of cowpea cultivars for resistance.
The reactions
4
of cowpea lines/cultivars to mechanical inoculation with five seed-
5
borne PTY+ isolates were determined (Table 6).
Five International
6
Institute of Tropical Agriculture (IITA) TW lines, TW-401, TVU-
7
408P2,
TW-1000, TW-1016-1, and TW-1582 and one U.S.A. cv White
8
Acre-BVR were immune to a11 isolates (i-e., asymptomatic and free
9
of ELISA MAb-detectable virus.
Some genotypes were susceptible to
10
a11 PTY+ isolates.
Other genotypes were susceptible to specific
11
isolates, e.g., cv Serido was susceptible only to PTY+ V17-14; cv
12
TVlJ-410 was susceptible only to PTY+ V54-23, whereas TW 984 was
13
resistant only to this isolate.
No attempt was made to classify
14
PTY+ plathotypes
using these
cowpea genotypes;
however,
the
15
genotypes provided evidence that the isolates were pathogenically
16
diverse.
17
Aplhid transmission.
Seed-borne PTY+ isolates Vl-1 and V17-14
18
were
both
efficiently
transmitted
nonpersistently by
Aphis
19
craccivura.
In replicated triais,,
isolate Vl-l was transmitted to
20
>60% (42 of 61) of the plants inoculated by 3 apterae/plant and, in
21
parallel tests, isolate V17-14 was transmitted to 59 of 59 plants
22
inoculated.
We believe that A. craccivora is a probable vector of
23
ail seed-borne potyviruses in Senegal and that indigenous biotypes
Plant Disease
Ndiaye, et a1
Page 12
13
1
cari transmit isolates of potyvirus PTY+ at rates comparable to
2
these experimental results.
3
DISCUSSION
4
Cowpea
viruses
are increasingly
important in
a11 cowpea
growing areas of Senegal.
The survey reported herein was prompted
because new pathogen/pest-resistant breeding lines had been damaged
by viral diseases.
Moreover, seed-borne viruses were designated
priority pathogens in these studies, since they have historically
9
inflict'ed heavy losses through unknowing establishment of seed-
10
borne field inoculum, followed by secondary spread by insect-vector
11
species (8).
12
The present study of 66 strategic cowpea samples with virus-
:13
1ik.e symptoms indicated the presence in Senegal of four recognized
:14
seed-borne viruses, CABMV, CSMV, CPMoV, and SBMV, and an apparently
15
new potyvirus herein designated PTY+.
SBMV had already been
16
reported from the Casamance regiom of Senegal (7).
Hampton et a1
1 7
(8), had detected CSMV in cowpea germplasm accessions from Senegal.
18
CPMoV was previously reported only from Nigeria (1, 18), until
19
recently when it was reported in Pakistan-grown cowpeas (2). CPMoV
20
was also detected in cowpea
sa'mples collected from screening
Plant Disease
Ndiaye, et a1
Page 13
----.. -.
--
.
-.
14
nurseries in Riverside, California, U.S.A (M. Bashir, unpublished
results).
The seed-borne nature of CPMoV (1, 18) and these recent
detections suggest that the virus is now spreading through seeds to
other parts of the world.
However, CABMV and potyvirus PTY+ were
the prevalent viruses, occurring in 83% of the 66 samples and
accounting for 55 of the 57 samples in which viruses were ELISA-
detected.
Based on prior investigations (8,
and unpublished
8
results), CMV and CPMV were expected to occur in Senegal-grown
9
cowpeas,
but neither was detected.
3.0
Multiple-virus infections tendto be common among samples from
11
field-grown cowpeas, world-wide.
Such mixed infections are known
12
to modify and complicate symptoms, essentially precluding field
1.3
diagnosis (9, 12).
In these studies, however, mixtures of seed-
14
borne viruses were found in only two of the 66 cowpea tissue
7.5
samples, CSMV + CPMoV and SBMV + CPMoV.
3.6
The five seed-borne PTY+ potyvirus isolates examined by DAS-
3.7
ELISA did not react with IgGs to CABMV and BlCMV (Table 3), a:nd
18
most CA:BMV and BlCMV isolates were non-reactive to IgG to PTY+
19
isolate V17-14 (Table 4).
Despite clear serological distinctions
;! 0
among these three viruses, the interactions of PTY+ isolates with
21
cowpea Igenotypes resembled CABMV, lacking the ability to infect
22
IITA accessions TVU-401 and TVU-1582, previously proven resistant
23
to a11 tested CABMV isolates (3, 4).
It is therefore conceivable
Plant Disease
Ndiaye, et a1
Page 14
c
15
1
that PTY+ is a distinct serotype and pathotype of CABMV and that
2
TVIJ-401 and TVU-1582 contain multiple genes/alleles against a11
3
tested CABMV variants.
4
While various controlmeasures may impede cowpea viraldisease
5
development, including control of insect vectors, removing diseased
6
plants from seed fields, and production of virus-free seed, we
7
believe the development of resistant cultivars
is the most
8
practical and economical control measure for such diseases. In
9
this situdy,
we identified six cowpea genotypes as
sources of
10
resistance to a11 isolates of PTYi-.
These genotypes have now been
11
incorporated in the extant Senegal cowpea breeding program for
12
development of
improved disease/pest-resistant
cultivars
for
13
Senegal cowpea production areas.
Plant Disease
Ndiaye, et a1
Page 15,
_-.-.
.-.
-7-
._
16
LITERA- CITED
1.
Allen, D.G., Thottappilly, G. and Rossel, H.W. 1982.
Cowpea
mottle virus: field resistance and seedtransmission in virus-
to:Lerant cowpea. Ann. Appl. Biol. 100:331-336.
2.
Bashir, M., and Hampton, R.O. X991.
Natural occurrence of five
cowpea
viruses in
Pakistan.
Phytopathology
81:1166-1167
(Abstr.) .
3.
Bashir, M., and Hampton, R. O.,
1991.
Blackeye cowpea mosaic
and
cowpea
aphidborne
mosaic
potyviruses:
Biological
comparisons
and serological
distinctions.
Phytopathology
81::L166 (Abstr.).
4.
Bashir, M.,
and
Hampton, R. 0.
1992.
Biological
characterization of' pathotypes of blackeye cowpea mosaic and
cowpea aphidborne mosaic potyviruses.
Phytopathology 82:1103
(Abstr.) .
5.
Clark, M.F. and Adams, A.N. 1977.
Characteristics of the
microplate method of enzyme-linked immunosorbent assay for the
detection of plant viruses. J., Gen. Virology. 34:475-483.
6.
Converse, R.H. and Martin, R.R.
1990.
ELISA methods for plant
viruses. In *‘
Serological
methods
for
detection
and
identification of viral and bacterial plant pathogens, A
laboratory manual. Hampton,R.O., Ball,S., and Boer, S. (Eds).
APS Press U.S.A.
389 pp.
7.
Gaikwad, D-G.
and Thottappilly, G.
1987.
Occurrence of
southern bean mosaic Virus on cowpea in
Senegal.
J.
Phytopathology. 120: 81-84.
8.
Hampton, R.O., Albrechtsen, S.E. and Mathur, S.B.
1992.
Seed
health (Viruses) of Viqna unsuiculata cultivars/selections from
developing countries.
Seed Science & Technol. 20:23-38.
9.
Har:rison, A.N. and Gudauskas, R.T.
1968.
Effects of some
viruses on growth and seed production of two cowpea cultivars.
Pla:nt Dis. Reptr. 52:509-511.
Plant Disease
Ndiaye, et a1
Page 16
c
17
1 0 .
Jensenius, J.C., Anderson, I., Hau, J., Crone, M., and Koch,
CI
1981.
Conveniently packaged antibodies.
Methods for
purification of yolk IgG.
Jour. of Immunol. Meth. 46:63-68.
1 1 .
Jordan, R.
1992.
Potyviruses,
monoclonal antibodies, and
antigenic sites.
Arch. Virol. (Suppl. 5):81-95.
12. Kuhn, C.W., Brantley, B.B. and G. Sowell, Jr.
1966.
Southern
pea viruses:
identification,
symptomatology and sources of
resistance.
Georgia Agric.
:Expt.
Stn.
Bull.
157,
Athens,
Georgia. 22 pp.
13. Lana, A.F.
and Adegbola,
M.O.K.
1977.
Important virus
diseases in West African crops. Rev. Plant Pathology 56:849-
865,
14. Lamptey, V-R. and Hamilton, B.L.
1974.
A new cowpea strain of
southern bean mosaic virus from Ghana.
Phytopathology 64:1100-
1104.
15. Mali, V.R. and Thottappilly, G.
1986.
Viruses on cowpea in
the tropics.
Rev. Tropical Plant Pathology 34: 421-522.
16. Ndiaye, M., Bashir, M., Keller, K. K., and Hampton, R. 0.
1992.
Identification, distribution and seed transmission of
cowpea viruses in Senegal.
Phytopathology 82:1103 (Abstr.).
17. Raheja, A.K. and Leleji, 0.1.
1974.
An aphid-borne mosaic
disease of irrigated cowpeas in northern Nigeria.
Plant
Disease 58: 1080-1084.
18. Shoyinka, S.A., Bozarth, R.F., Reese, J. and Rossel, H.W.
1978 a
Cowpeas
mottle
virus: A
seed-borne
virus
with
distinctive
properties
infecting
cowpea in
Nigeria.
Phytopathology 68:693-699.
19.
Thiaw, S., Hall, A.E.,
and Parker, D.R.
1993.
Varietal
intercropping andthe yields and stability of cowpea production
in semiarid Senegal.
Field Crops Research (in press).
20. Thottappilly, G. and Rossel, H.W.
1985.
World occurrence and
distribution of virus diseases.
In Cowpea Research, production
and Utilization.
Singh, S.R., Rachie, K.O. (Eds.). John Wiley
& Sons, Chichester, Great Britain, London.
Plant Disease
Ndiaye, et a1
Page 17
._----”- .--- --_“-
f
18
21. Wang, W., Mink, G. I., Silbernagel, M. J., and Davis, W. 12.
1984.
Production
of hybridoma
lines
secreting
specific
antibodies to bean common mosaic virus
(BCW
strains,
Phytopathology 74:1142. (Abstr.).
22. WillLiams, R. J.
1975.
Diseases of cowpea [ Vigna unguiculaiza
(L.) Walp. ] in Nigeria. PANS 21~253-257.
Plant Disease
Ndiaye, et a1
Page 18
.--_
--
-- . . ..-
Table 1.
Viruses detected by DAC-ELISA in field samples of cowpea collected in
five districts of Senegal (West Africa)
No. of samples reacting positively with antiviral
antisera/potyvirus MAb.
District
No.
-------------------------------------------------------------
surveyed/
samples
BlCMV CABMV CMV
CPMoV
CPMV
CSMY
SBMV
MAB
sampled
collected
II-197
Diourbel
28
- 1
10
-
1
1
11 2
Kolda
4
1
-
2
Louga
20
10
-
1
2
1
7
Tambacounda 10
9
-
1
Thies
4
4
-
Total
66
34
-
2
1
1
21
1
-
I
indicates virus not detected by ELISA.
2
Samples reacting to monoclonal antibody II-197 (21) contained no virus
detectable by other anti-viral polyclonal antisera.
Plant Disease
Ndiaye, et a1
Page 19
.
.J
<,;yr:.r
-.-i.,,
--------------------_I___________)c_cc__-------------~-~--~~--~--------------~--
Table 2.
Disease incidence' and seed transmission2 associated with Senegal
potyvirus PTY+ field isolates
Disease
No. of seeds
Seed
Virus
Cultivars/
incidence
germinated/
transmission
isolate lines
(%)
planted
Incidence %
v 1
Baye Ngagne
100
37/50
1137
3
IS86-275N
61
41/50
0/41
0
IS86-283-15
8
29/50
0/29
0
Mougne
97
46/50
2146
4
58-57
100
46150
6146
13
v 2
Baye Ngagne
100
46/100
0/46
0
IS86-275N
47
37/100
0137
0
1686-283-15
18
44/100
0144
0
Mougne
100
921100
1192
1
58-57
55
831100
7183
8
v 17
Baye Ngagne
81
035/50
0/35
0
IS86-275N
50
40/50
2/40
5
1886-283-15
52
23/50
0/23
0
Mougne
97
47150
0147
0
58-57
93
43/50
13/43
30
v 54
Baye Ngagne
86
27/50
0127
0
IS86-275N
88
40/50
2/40
5
1586-283-15
82
28150
0/28
0
Mougne
97
49/50
0/49
0
58-57
89
44/50
1/44
2
' Experiments conducted in field and screenhouse plots, Bambey, Senegal.
2
Seeds taken from potyvirus inoculated plants, Bambey screenhouse plots.
Experiments
conducted in greenhouses, Corvallis, OR, U.S.A.
0
Plant Disease
Ndiaye, et a1
Page 20
Table 3.
CCRilFiXiSQnS ^f eight B2CîrJniZed F@tyV iruses with five seed-borne Senegal
cowpea potyvirus isolates, by DAS- and DAC-ELISA.
The Senegal isolates were also
tested for the possible presence of four seed-borne non-potyviruses
A 4O5 values '
Seed-borne isolates of Senegal potyvirus, PTY+
Homolo-
Healty-
gous
plant
Antiserum
Vl-1
V17-2
v17-14
v54-3
V54-23
virus 2
extract
DAS-ELISA
BlCMV 3
0.00
0.01
0.01
0.00
0.01
0.95
0.01
CABMV
0.01
0.01
0.01
0.01
0.03
1.15
0.01
PSbMV
0.00
0.00
0.00
0.00
0.00
1.87
0.00
CM-V
0.01
0.00
0.01
0.00
0.00
1.27
0.02
DAC-ELISA
BlCMV
0.26
1.20
0.36
0.37
0.21
2.81
0.02
CABMV
1.68
1.65
1.51
1.30
1.41
2.48
0.02
CYW
0.11
0.10
0.08
0.05
0.08
>3.00
0.01
PeMoV
1.63
1.38
2.03
1.27
1.58
>3.00
0.03
Pstv
1.90
1.50
1.75
1.10
1.92
>3.00
0.02
PM-V
1.86
0.54
0.23
0.78
0.51
>3.00
0.02
PSbMV
1.65
1.58
1.51
1.86
2.11
>3.00
0.01
WLMV
0.01
0.02
0.01
0.02
0.04
>3.00
0.01
CPMoV
0.03
0.02
0.02
0.02
0.00
>3,00
0.03
CSMV
0.16
0.10
0.11
0.15
0.13
>3.00
0.12
SBMV
0.00
0.00
0.00
0.00
0.02
>3.00
0.01
II-197
0.31
0.77
1.40
0.57
0.70
0.58 4
0.00
(BCMV MAb)
Agdia
0.22
NT 5
0.70
0.78
0.86
0.77 6
0.00
(PTY MAb)
Plant Disease
Ndiaye, et a1
Page 21
Table 3; cent.
1
Ado values recorded after 90 min incubation with substrate, p-nitrophenyl
phosphate.
2
Virus homologous to each antiserum, e.g., the viral homologue of BlCMV
antiserum is blackeye cowpea mosaic potyvirus.
3
Antisera to potyviruses are:
BlCMV, blackeye cowpea mosaic virus; CABMV,
cowpea aphid-borne mosaic virus; CYW, clover yellow vein virus; PMV, pea
mosaic virus, PeMoV, peanut mottle virus; PSbMV, pea seedborne mosaic virus;
PStV, peanut stripe virus; and WLMV, white lupin mosaic virus.
Antisera
to other viruses seed-borne in cowpea are:
CMV, cucumber mosaic virus; CSMV,
cowpea severe mosaic virus; SBMV, southern bean mosaic virus; and CPMoV, cowpea
mottle virus.
The Agdia monoclonal antibody (11) reacts to >90% of a11 tested
potyviruses; monoclonal antibody II-197 (21), produced against bean common
mosaic virus, reacts to its homologue and several other potyviruses.
4
BlCMV isolate RF-26B was selected as a positive control for MAb 11-197.
5
NT = Not tested.
6 CABMV isolate 9-7C was selected as a positive control for the Agdia PTY MAb.
Plant Diesease
Ndiaye, et a1
Page 22
23
Table 4.
DAS-ELISA tests of selected BlCMV and CABMV
isolates against chicken anti-V17-14 immuno-
gammaglobulin G
-7
Virus
isolate
A= values
B:LCMV-Ga '
0.003
PI-3B
0.005
RF-4B
0.012
PU-7B
0.004
PIJ-8B
0.003
PI-22B
0.007
PIC-23B
0.002
PI-25B
0.003
RI?-26B
0.002
RF-27B
0.007
RN-7c ’
RN-1oc
2
RN-27C
RN-28C
RN-34c
RN-35c
RN-36C
RN-37c
PI-39c
PI-4oc
PI-44C6
V:L7-14 (Homologue)
Healthy-plant
extract
' Isolates BlCMV-Ga (Georgia) and RN-7C (Botswana) were
included as type isolates of BlCMV and CABMV,
respectively.
2
Isolates RN-27C and RN--28C previously had reacted in
DAS-ELISA only to CABAN antiserum produced against
CABMV isolate RN-7C.
Plant Disease
Ncliaye, et a1
Pa&ge 23
-. _._------
Table 5.
Reactions by selected plant species to inoculations with five seed-borne
isolates of Senegal potyvirus PTY+
Disease reactions to PTY+ isolates
Host species
VI-1
W-7-2
v17-14 v54-3
V54-23
Lequminous hosts:
Lupinus albus cv Astra
- 1
Medicago sativa cv DuPuits
Trifolium pratense cv Kenland
Phaseolus vulgaris cv Monroe
Phaseolus vulgaris cv Top Crop
Vicia faba CV Hertz Freya
VN
VN
Non-lequminous hosts:
Chenopodium amaranticolor,
LLn
LLn.VN LLn
LLn,VN
Corvallis strain
Nicotiana benthamiana, ATCC
SM
S M
SM
S M
SM
Gomphrena globosa, A.F. Ross Strain
Phlox drumondii cv Ta11 Mixed Color
Lycopersicon esculentum cv Marglobe
Petunia hybrida cv King Henry
Antirrhinum majus cv Mixed Colors
1
Symbols are: - , no symptoms and no ELISA-detectable virus; LI, latent
(asymptomatic), ELISA-detectedinfection; LLn, necrotic locallesions; VN, vein
necrosis; SM, systemic mosaic.
2
Plant Disease
bidiaye, et a1
Page 24
25
Table 6.
Responses of selected cowpea genotypes to green-
house mechanical inoculations with five seed-
borne isolates of Senegal potyvirus PTY+
-
Potyvirus isolates
C o w p e a
genotypes
Vl-1
V17-2
v17-14
v54-3
V54-23
TVU 109P2 '
+ +
T V U 1 9 6
++
Tvu 347
+ +
++
Tvu 354
+ +
++
Tvu 401
TVU 408P2
Tw 410
TVU 984
+ +
++
TV-U 1000
TVU 1016-L
TVU 1582
TVU 2657
.
+ +
++
Tvu 3433
+ +
++
IT 81D 1137
+ +
++
IT 86 27N
+ +
++
P:I 25122
+ +
++
Bambey 21
++
Serido
Wlh Acre BVR
Cal Bl # 5
0
++
Snapper
0
++
Blue Goose
0
Corona
0
Mlopod
0
Tex Cr # 8
0
Tlex Cr # 40
0
UCR 524B
0
Mis Purple
0
Mis Silver
0
Magnolia
0
K.n Pur Hull
0
Worthmore
0
Bettergreen
0
UCR 7964
0
1
TW genotypes kindly provided by I.I.T.A., Ibadan, Nigeria.
‘2
Symbols are: - , no symptoms and no virus detectable by ELISA,
.1-e., immune; + , mild systemic symptoms; ++, moderate systemic
symptoms; LI, latent (asymptomatic),
ELISA-detected infection;
i-e.,
tolerant to infection; 0, not tested.
Plant Disease
Ndiaye, et a1
Page 25