1 23 Archives of Virology Official Journal of the Virology Division of the International Union of Microbiological Societies ISSN 0304-8608 Arch Virol DOI 10.1007/s00705-014-2260-7 Mapping the distribution of maize streak virus genotypes across the forest and transition zones of Ghana Allen Oppong, Samuel K. Offei, Kwadwo Ofori, Hans Adu-Dapaah, Joseph N. L. Lamptey, Brigitta Kurenbach, Matthew Walters, et al.
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1 23
Archives of VirologyOfficial Journal of the VirologyDivision of the International Union ofMicrobiological Societies ISSN 0304-8608 Arch VirolDOI 10.1007/s00705-014-2260-7
Mapping the distribution of maize streakvirus genotypes across the forest andtransition zones of Ghana
Allen Oppong, Samuel K. Offei, KwadwoOfori, Hans Adu-Dapaah, JosephN. L. Lamptey, Brigitta Kurenbach,Matthew Walters, et al.
1 23
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BRIEF REPORT
Mapping the distribution of maize streak virus genotypesacross the forest and transition zones of Ghana
Allen Oppong • Samuel K. Offei • Kwadwo Ofori • Hans Adu-Dapaah •
Joseph N. L. Lamptey • Brigitta Kurenbach • Matthew Walters • Dionne N. Shepherd •
Darren P. Martin • Arvind Varsani
Received: 20 May 2014 / Accepted: 15 October 2014
� Springer-Verlag Wien 2014
Abstract Throughout sub-Saharan Africa, maize streak
virus strain A (MSV-A), the causal agent of maize streak
disease (MSD), is an important biological constraint on
maize production. In November/December 2010, an MSD
survey was carried out in the forest and transition zones of
Ghana in order to obtain MSV-A virulence sources for the
development of MSD-resistant maize genotypes with
agronomic properties suitable for these regions. In 79 well-
distributed maize fields, the mean MSD incidence was
18.544 % and the symptom severity score was 2.956
(1 = no symptoms and 5 = extremely severe). We detec-
ted no correlation between these two variables. Phyloge-
netic analysis of cloned MSV-A isolates that were fully
sequenced from samples collected in 51 of these fields,
together with those sampled from various other parts of
Africa, indicated that all of the Ghanaian isolates occurred
within a broader cluster of West African isolates, all
belonging to the highly virulent MSV-A1 subtype. Besides
being the first report of a systematic MSV survey in Ghana,
this study is the first to characterize the full-genome
sequences of Ghanaian MSV isolates. The 51 genome
sequences determined here will additionally be a valuable
resource for the rational selection of representative MSV-A
variant panels for MSD resistance screening.
Keywords Maize streak virus � Genomic � Survey �Maize streak disease � Symptoms � Ghana
Maize streak virus (MSV), belonging to the genus Mas-
trevirus of the family Geminiviridae [12], is the most
damaging viral pathogen of maize (Zea mays L.) in Africa
[25]. It is the causal agent of maize streak disease (MSD),
which is endemic throughout sub-Saharan Africa and the
adjacent Indian Ocean islands [2, 17, 21]. MSV is a single-
stranded circular DNA virus with a *2.7-kb genome that
GenBank Accession numbers: KJ699303–KJ699353.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00705-014-2260-7) contains supplementarymaterial, which is available to authorized users.
A. Oppong (&) � H. Adu-Dapaah � J. N. L. Lamptey
CSIR-Crops Research Institute, P.O. Box 3785, Kumasi, Ghana
e-mail: [email protected]
S. K. Offei � K. Ofori
West Africa Centre for Crop Improvement, College of
Agriculture and Consumer Science, University of Ghana,
P.O. Box 30, Legon, Ghana
B. Kurenbach � M. Walters � A. Varsani (&)
School of Biological Sciences, University of Canterbury, Private
Bag 4800, Christchurch, New Zealand
e-mail: [email protected]
D. N. Shepherd
Department of Molecular and Cell Biology, University of Cape
Town, Rondebosch, Cape Town 7701, South Africa
D. P. Martin
Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Anzio Road, Observatory,
Cape Town 7925, South Africa
A. Varsani
Biomolecular Interaction Centre, University of Canterbury,
Private Bag 4800, Christchurch, New Zealand
A. Varsani
Department of Plant Pathology and Emerging Pathogens
Institute, University of Florida, Gainesville, FL 32611, USA
A. Varsani
Electron Microscope Unit, Division of Medical Biochemistry,
Department of Clinical Laboratory Sciences, University of Cape
Town, Rondebosch, Cape Town 7701, South Africa
123
Arch Virol
DOI 10.1007/s00705-014-2260-7
Author's personal copy
is encapsidated within geminate particles [2, 6] and is
transmitted by leafhoppers of the genus Cicadulina [23].
Infection with MSV causes severe chlorosis on newly
emerged leaves of MSV-susceptible maize cultivars,
leading to stunted growth, poor ear formation, reduced seed
setting, and, when plants are infected at a young age, heavy
yield losses or premature death [4, 10].
Eleven genetically distinct MSV strains have been
identified (referred to as MSV-A through MSV-K [9, 10,
18, 26, 27]) with varying degrees of virulence in maize [9].
However, only one, MSV-A, is known to cause economi-
cally significant MSD [20]. This strain can be found
infecting and causing severe yield losses in maize grown in
forest and savanna zones from sea level up to 1800 m [1].
MSV-A variants have been genetically categorized into
five distinct subtypes, named MSV-A1, MSV-A2, MSV-A3,
MSV-A4, and MSV-A6 [9, 26]. Whereas MSV-A1 is found
throughout sub-Saharan Africa, MSV-A2, MSV-A3, MSV-
A4, and MSV-A6 have only ever been found in West
Africa, East Africa, southern Africa and the Indian Ocean
islands, respectively [11]. The virulence in maize of dif-
ferent MSV-A isolates can vary quite broadly [20]. For
example, the MSV-A3 variants ‘‘Nigeria mild’’ (MSV-A3
[NG-Nm-1983]) and ‘‘Nigeria severe’’ (MSV-A3 [NG-Ns-
1983]) differ at only three nucleotide sites but induce
markedly different degrees of chlorosis, streak widths, and
streak lengths [3].
Although large numbers of MSV-resistant maize geno-
types already exist, there remains a continuing need to
develop additional high-yielding resistant genotypes that
are tailored to each of Africa’s diverse maize-growing
environments. Amongst these environments are the tropical
forest and transition zones of Ghana, where, despite a
pressing need for locally adapted MSV-resistant maize
genotypes [22], almost nothing is presently known about
either the presence or distribution of the various different
MSV-A genotypes. A key component of future Ghanaian
maize-breeding efforts should be the consistent use of
standardized panels of local viral genotypes to select for
MSV resistance. Towards this end, the objectives of this
study were to conduct the first-ever Ghanaian MSD survey,
to identify MSV strains found in the forest and transition
zones of this country and to secure MSV inoculum sources
for the breeding of novel MSV-resistant maize genotypes.
In November/December (during the minor maize-
growing season) of 2010, a MSV disease survey was
conducted in the main maize-growing areas of Ghana,
including forest and transition zones of Brong Ahafo,
Ashanti, the eastern region, and parts of the central region.
Forest zones occur in the south-central parts of Ghana, a
region with semi-deciduous forests and an annual rainfall
of 1200-1600 mm (mean, 1500 mm). The transition zones
with semi-deciduous forests transitioning into Guinea
savannah vegetation cover the more northerly parts, mainly
in the northern Ashanti and Brong Ahafo regions of Ghana,
and have an annual rainfall of 1100-1400 mm (average,
1300 mm). Both the forest and transition zones experience
two periods of increased rainfall between March and July
and between September and November. Seventy-nine
farms were visited, the specific locations of which were
captured with a global positioning system device (GPS;
Table 1 and Fig. 1). Farms were greater than five kilome-
ters apart. Transect walks were made in each farm, and
samples were collected from selected plants showing dis-
ease symptoms. Each sample collected was scored for
disease severity based on a 1–5 scale adopted from Kye-
tere, et al. [7] (Supplementary Figure 1) with a modifica-
tion of 0.5 increments; where 1 represents no infection; 2,
mild infection; 3, moderate infection; 4, severe infection;
and 5, very severe infection. Disease incidence per field
was estimated as the percentage of plants along the tran-
sects displaying MSD symptoms (Fig. 1). From each farm,
three to five samples were collected, labeled and stored by
pressing on paper for further processing.
Mean virus disease incidence across the study areas was
18.544 %, with the highest incidence in an infected field
being 50 % and the lowest in an infected field being 5 %
(Table 1; Fig. 1). The mean disease severity score across
all MSV-infected fields was 3.009 (SD = 0.694) with the
means for individual fields ranging from 2.0 to 4.1 (Sup-
plementary Table 1; Fig. 1).
Overall MSD incidence in the transition zone (20.278 %)
was similar to that observed in the forest zone (18.033 %)
(Fig. 1) and so was the MSD severity in the transition zone
(mean score = 3.074; SD = 0.625) and the forest zone
(mean score = 2.993; SD = 0.757). The mean MSD inci-
dence for maize varieties reported to have improved MSV
resistance was 13.733 %, with a mean severity score of 2.754
(SD = 0.691), whilst the MSD incidence for local ‘‘unim-
proved’’ varieties was 19.672 %, with a mean severity score
of 3.070 (SD = 0.730). Although these differences between
the MSD-resistant and unimproved varieties were significant
for both the incidence (0.000326; 2-tailed Mann-Whitney
U-test) and severity scores (p = 0.02726; 2-tailed Mann-
Whitney U-test), across all of the 79 farms, we detected no
correlation between MSD incidence and MSD severity
(Pearson’s R = -0.08).
We noted, however, that whereas MSD-resistant maize
genotypes were grown in only 11 % of the fields sampled
in the transition zones, in the forest zones, 21 % of sampled
fields were under MSD-resistant maize. It was plausible,
therefore, that the apparently lower MSD incidences and
severity scores determined for the MSD-resistant maize
genotypes might be attributable to differences in the cli-
mates between the two ecological zones. However, when
we focused on MSD incidence and symptom severity
A. Oppong et al.
123
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Maize streak virus genotypes in Ghana
123
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A. Oppong et al.
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33
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0.9
13
0-
-
13
56
.16
8-
0.1
89
FU
43
2.5
33
3.1
00
.55
15
MS
V-A
[GH
-gh
13
5-
Ab
a-2
010
]K
J69
93
22
13
65
.86
5-
0.6
7F
U2
32
.52
.52
.52
.50
0.3
52
5M
SV
-A[G
H-g
h1
36
-A
si-2
01
0]
KJ6
993
23
13
76
.29
1-
0.4
76
FU
1.5
2.5
22
.00
0.5
02
5M
SV
-A[G
H-g
h1
37
-A
hy
-201
0]
KJ6
993
24
13
86
.38
3-
0.5
52
FU
2.5
34
24
.53
.20
1.0
42
4-
-
13
96
.71
2-
1.5
23
FU
44
4.5
44
4.1
00
.22
15
MS
V-A
[GH
-gh
13
9-
Fu
m-2
01
0]
KJ6
993
25
14
06
.69
9-
1.6
53
FU
4.5
3.5
44
44
.00
0.3
52
5M
SV
-A[G
H-g
h1
40
-K
wa-
20
10
]K
J69
93
27
14
16
.96
7-
1.6
83
FU
34
4.5
33
3.5
00
.71
20
MS
V-A
[GH
-gh
14
1-
Nam
-2010]
KJ6
993
28
14
27
.07
2-
1.7
17
FU
2.5
33
.53
.00
0.5
02
1M
SV
-A[G
H-g
h1
42
-A
br-
20
10]
KJ6
993
29
14
37
.23
1-
1.8
06
FU
44
33
3.5
3.5
00
.50
21
MS
V-A
[GH
-gh
14
3-
Ase
-20
10]
KJ6
993
30
14
47
.28
6-
1.8
55
FU
3.5
4.5
33
3.5
00
.71
18
MS
V-A
[GH
-gh
14
4-
Daa
-20
10
]K
J69
93
31
14
57
.31
8-
1.9
05
FU
33
.53
2.5
33
.00
0.3
51
9-
-
14
67
.40
9-
1.9
73
FR
33
3.5
2.5
3.5
3.1
00
.42
10
MS
V-A
[GH
-gh
14
6-
Afr
-201
0]
KJ6
993
32
14
77
.39
6-
1.9
59
FU
22
3.5
2.5
2.5
00
.71
20
--
14
87
.30
6-
2.0
74
TU
3.5
44
33
3.5
00
.50
22
MS
V-A
[GH
-gh
14
8-
Su
r-2
01
0]
KJ6
993
33
14
97
.27
4-
2.0
39
FU
1.5
2.5
22
2.5
2.1
00
.42
22
--
15
06
.91
7-
1.9
03
FU
2.5
23
32
2.5
00
.50
15
--
15
16
.90
7-
1.8
86
FU
2.5
2.5
2.5
2.5
00
.00
17
MS
V-A
[GH
-gh
15
1-
Po
t-2
010
]K
J69
93
34
15
26
.81
7-
1.8
62
FU
22
22
.00
0.0
01
8-
-
15
36
.76
8-
1.7
86
FR
34
32
33
.00
0.7
18
MS
V-A
[GH
-gh
15
3-
To
p-2
01
0]
KJ6
993
35
15
46
.63
5-
1.8
1F
R3
43
.53
.50
0.5
05
MS
V-A
[GH
-gh
15
4-
Nk
a-2
010
]K
J69
93
36
15
56
.63
5-
1.9
45
FU
43
3.5
43
3.5
00
.50
10
MS
V-A
[GH
-gh
15
5-
Kw
a-20
10
]K
J69
93
37
Maize streak virus genotypes in Ghana
123
Author's personal copy
scores of the unimproved maize varieties (for which suf-
ficiently large samples were available from both ecological
zones), we failed to detect any significant associations
between these and the ecological zone within which fields
were situated (p-values, 0.41 and 0.74 for the incidence and
severity scores, respectively; 2-tailed Mann-Whitney
U-test). This suggests that the lower degrees of MSD
incidence and severity in the MSD-resistant maize varieties
are indeed attributable to their inbred resistance rather than
to the fact that they appear to be used more routinely in the
forest zones than in the transition zones.
Fifty-four leaf samples were randomly taken from the
collection of dried leaf samples, and total DNA was
extracted using an Epoch nucleic acid purification kit
according to the manufacturer’s instructions (Epoch Life
Science, Inc, USA). Full MSV circular genomes were
enriched using phi29 DNA polymerase (TempliPhiTM, GE
Healthcare, USA) as described by Owor et al. [15] and
Shepherd et al. [19]. Concatenated viral genomes were
then digested using the restriction enzyme BamHI to yield
linearized genomes (*2.7 kb). The fragments were
resolved using 0.7 % agarose gels, and fragments of 2.7 kb
were gel purified using an Intron Gel Purification Kit
(according to the manufacturer’s instructions; Intron,
Korea) and then ligated to the BamHI site of the
pGEM3Zf ? vector (Promega Biotech, USA). The result-
ing clones were sequenced by Macrogen Inc. (Korea) by
primer walking. Viral genomes were assembled using
DNAMAN (version 7; Lynnon Biosoft). From these 54
samples, 51 Ghanaian MSV-A genomes were recovered,
and the remaining three were sub-full-genome length
(subgenomic) MSV-A sequences with deletions of various
sizes. The 51 full genomes, together with all other genomes
of MSV-A available in GenBank, were aligned using
MUSCLE [5] with default settings implemented in MEGA
5 [24]. Phylogenetic analysis was conducted using MEGA,
by the neighbor-joining tree method with the (Jukes-Can-
tor) nucleotide substitution model and branch support tes-
ted with 1000 bootstrap replicates. The degree of similarity
among strains was calculated using SDT v 1.0 [12].
Phylogenetic analysis of the 51 Ghanaian MSV-A full
genomes together with other MSV-A isolates sampled from
elsewhere in Africa indicated that all of the Ghanaian
viruses belonged to the virulent MSV-A1 subtype (Fig. 2).
All of the Ghanaian isolates were also clearly clustered
within clades primarily containing other West-African
MSV-A1 sequences (from Burkina Faso [BF], Benin [BJ],
Nigeria [NG] and Cameroon [CM]).
The Ghanaian isolates did not show any obvious clus-
tering by either geographical location or ecological zone,
with isolates obtained from the transition and forest zones
and from widely separated parts of the country intermin-
gling within the phylogenetic tree. For instance, isolateTa
ble
1co
nti
nu
ed
Sam
ple
IDL
atL
on
Tra
nsi
tio
n(T
)/
fore
st(F
)
Mai
zev
arie
tyre
sist
ant
(R)
/u
nim
pro
ved
(U)
Sam
ple
1S
amp
le2
Sam
ple
3S
amp
le4
Sam
ple
5M
ean
sym
pto
mse
ver
ity
sco
re
Std
dev
iati
on
%M
SV
inci
den
cein
fiel
d
Gen
Ban
kID
Gen
Ban
kac
cess
ion
#
15
66
.59
8-
2.1
19
FU
22
.52
.53
.52
2.5
00
.61
22
--
15
76
.61
9-
1.8
56
FR
22
22
.00
0.0
01
2M
SV
-A[G
H-g
h1
57
-S
ei-2
01
0]
KJ6
993
38
15
86
.50
8-
1.8
53
FU
2.5
2.5
2.5
2.5
00
.00
22
--
15
96
.50
8-
2.0
83
FU
33
42
.53
3.1
00
.55
21
MS
V-A
[GH
-gh
15
9-
Yam
-2010]
KJ6
993
39
16
06
.45
3-
1.5
7F
R3
.52
.53
3.5
33
.10
0.4
21
5-
-
16
16
.69
2-
1.4
34
FU
32
23
2.5
00
.58
24
--
16
26
.63
1-
1.4
51
FU
3.5
3.5
33
.54
3.5
00
.35
22
MS
V-A
[GH
-gh
16
2-
Kw
a-20
10
]K
J69
93
40
Gen
Ban
kac
cess
ion
num
ber
sfr
om
whic
hM
SV
-Agen
om
esw
ere
reco
ver
edar
eal
sopro
vid
ed
A. Oppong et al.
123
Author's personal copy
MSV-A [GH-gh114-Hia-2010] (KJ699311) from the tran-
sition zone and isolate MSV-A [GH-gh155-Kwa-2010]
(KJ699337) from the forest zone formed one of the only
supported Ghanaian MSV-A clusters in this tree (with
93 % bootstrap support).
Similarly, disease severity scores estimated for sampled
leaves did not show any obvious association with the
phylogenetic clustering of isolates. For instance, leaves
from the farms where MSV-A [GH-gh29-Nsa-2010]
(KJ699344) and MSV-A [GH-gh162-Kwa-2010]
(KJ699340) were isolated had an average symptom
severity of 2.5 and 3.5, respectively, and the recovered
genomes from the two plants obtained from these two
farms had 99.6 % genome-wide nucleotide sequence
identity. On the other hand, MSV isolates MSV-A [GH-
gh123-Bes-2010] (KJ699315) and MSV-A [GH-gh144-
Accra
CÔTED'IVOIRE GHANA
Gulf of Guinea
0 50
0 50
miles
km N
113109
112
110111
106108105142
143141
151150
117116
101 118
103102104 119
107123
115
120
122 121124
161139
140153
152
162157154155
156
159 158125
160
126127
128
129 130131
132 133136
134
137138
135
5758
5655
5253
59
54
3 131
114
1924
2932
22
147146
144145
149148
2
1–10
11–2
021
–30
41–5
03
4
% Incidence
MSV
Seve
rity
(1–5
)
Kumasi
Accra
BENIN
BURKINA FASO
CÔTED'IVOIRE
GHANA
TOGO
Fig. 1 Mapped MSV sampling locations with symptom severity and incidence index. Transition zone sampling sites are highlighted in red
Maize streak virus genotypes in Ghana
123
Author's personal copy
Daa-2010] (KJ699331), from fields that both had an aver-
age symptom severity score of 3.5, shared 99.3 % genome-
wide nucleotide sequence identity (Fig. 2).
Amongst all eleven of the known MSV strains, only
MSV-A causes severe MSD [26]. Here, for the first time,
we have both surveyed the incidence of MSD and assessed
the diversity of MSV-A variants in the important maize-
growing transition and forest zones of Ghana.
The MSD symptom severity and incidence data indi-
cated the extent and severity of MSD in the 2010 minor
maize-growing season across the main maize-growing
regions of Ghana. Assuming an average yield loss of 50 %
per infected plant, the observed MSD incidences could
result in an average yield loss of 7 % in fields where maize
varieties with certified MSD resistance are grown (which
experience a 13.733 % MSD incidence) and 10 % in fields
KJ69
9321
KJ69
9333
KJ69
9310
KJ69
9312
KJ69
9335
KJ69
9305
KJ69
9332
KJ69
9328
KJ69
9331
KJ69
9306
KJ69
9322
KJ69
9352
KJ69
9339
KJ699341
KJ699347
KJ699345
KJ699350
KJ699330
KJ699309
KJ699314
KJ699344
KJ699340
KJ699337
KJ699327KJ699313
KJ699346KJ699343
KJ699311KJ699334
KJ699349
KJ699320
KJ699342
KJ699308KJ699338
623996JK KJ69
9351
KJ69
9316
KJ69
9319
KJ69
9336
KJ69
9304
KJ69
9325
KJ69
9323
KJ69
9324
KJ69
9317
KJ69935
3
KJ699315
KJ699307
KJ699303
KJ699329
KJ699318
KJ699348
9086
77 71
91
66 95
77
8966
8599
86
99
99
84
86
98
89
62
6572
89
9376
7998
7472
63
99
100
81
HQ69
3377
HQ69
3387
HQ69
3392
HQ69
3282
HQ69
3285
763396QHHQ69
3368
HQ69
3371
EU62
8566
HQ69
3381
HQ69
3327
KJ43
7664
HQ6933
84
HQ693372
HQ693373
HQ693390
HQ693389HQ693323HQ693324
HQ693302HQ693321HQ693379
HQ693385HQ693286FJ882089
HQ693306
HQ693386KJ437663HQ693320
HQ693322
HQ693298
HQ693305
HQ693369
KJ437657
HQ693376
HQ693378
EU628567
HQ693318KJ437661
HQ693281
HQ693304 KJ43
7662
HQ69
3326
HQ69
3325
KJ43
7658
HQ6933
91HQ693
308HQ693
370
HQ693380
HQ693309
HQ693383
HQ693328
HQ693374
HQ693382
HQ693375
FM210279
HQ693393
HQ693319KJ437659KJ437660
HQ693316EF547091
AF329878EF547102EF547114EF547083EF547121AF329885FJ882091X01089 FJ882146X01633
FJ882106
0.002
21–
1011
–20
21–3
041
–50
3
4
GhanaBurkina FasoBeninNigeriaCameroonChadCentral African RepublicUgandaKenyaZimbabwe
% Incidence
MSVSe
verit
y (1
–5)
Fig. 2 Neighbor-joining phylogenetic tree of MSV-A isolates from
this study together with closely related viruses from other African
countries. Numbers associated with tree branches represent the
percentage of 1000 bootstrap iterations supporting these branches.
Branches with less than 60 % bootstrap support have been collapsed.
Transition zone sampling sites are highlighted in red
A. Oppong et al.
123
Author's personal copy
where varieties without certified resistance are grown
(which experience a 19.672 % MSD incidence). Given the
fact that most Ghanaian maize farmers rely more heavily
on the use of varieties without any reported MSD resis-
tance (81 % of the fields sampled here, and see [16]), it is
likely that, on a country-wide scale, yield losses due to
MSD would more closely approach those encountered by
the unimproved varieties. While these findings starkly
underline the urgent need in Ghana to develop genotypes
with increased resistance to the currently circulating MSV
variants, they also stress the need for further studies into
why currently available MSD-resistant maize genotypes
are not more widely used in the country.
Surprisingly, of the 51 full MSV-A genomes determined
here, all belonged to the MSV-A1 subtype. MSV-A1 is the
only MSV-A subtype that has a Pan-African distribution,
with subtypes MSV-A2, MSV-A3, MSV-A4, and MSV-A6
only ever having been found in West Africa, East Africa,
Southern Africa and the Indian Ocean islands, respectively
[11]. One of the isolates examined here, MSV-A [GH-
gh58-Nya-2010] (KJ699352), actually holds the distinction
of being the most westerly MSV-A1 isolate ever identified
and, as such, its sampling location demarcates the known
western limit of the geographical distribution of MSV-A1.
The only other MSV-A subtype that has ever been
detected in West Africa is MSV-A2. This subtype has not,
however, been detected in this region since 1987. Given
that 102 West African MSV-A [3, 8, 11, 13] isolates have
been sampled and characterized since 1990 (including the
51 presented here for the first time), the absence of MSV-
A2 isolates amongst these suggests that this subtype may be
extinct. Similar apparent disappearances of subtype MSV-
A3 from East Africa (the last recorded sample was isolated
in 1997; Monjane et al. [11]) and subtype MSV-A6 from
Reunion Island (which has remained undetected since 1995
despite repeated MSV sampling efforts carried out on the
island since 2006; unpublished data) indicate that this is not
a unique occurrence.
A large-scale time-calibrated phylogeographic survey of
African MSV-A diversity has indicated that all of the
known MSV-A1 lineages that are presently circulating in
West Africa likely arose in East Africa within the past
40 years [11]. However, very little information is available
on the movements of MSV-A variants within West Africa.
Although we have found phylogenetic evidence of wide-
spread epidemiological mixing of MSV-A1 variants
between the different West African countries (i.e., the
Ghanaian isolates are not all in a monophyletic clade), it
remains uncertain whether these movements have been
from Ghana to the rest of the West African countries or
vice versa (supplementary Figure 2). Such movements will
be particularly difficult to infer using phylogenetic
approaches because of the absence of clear phylogenetic
clustering of Ghanaian MSV-A genotypes. Although MSV-
A genotypes display detectable degrees of phylogenetic
clustering at the continental scale, MSV surveys in other
parts of Africa have also revealed that such clustering is
generally not as evident at the scale of individual countries
[11, 14].
It should be stressed that although we failed to detect
any association between MSD symptoms/incidences in
individual fields and the phylogenetic clustering of viruses
isolated from these fields, this does not indicate that there is
no association between virus genotype and virulence. It is,
in fact, entirely plausible that individual fields (and possi-
bly even individual sampled plants) that were examined in
this study could have been infected by multiple genetically
distinct MSV genotypes. For example, in a similar MSD
survey carried out in Uganda in 2005, mixed infections
involving genetically distinct MSV lineages were evident
in 66 % of sampled fields (and in 6.25 % of individual
sampled leaves [14]). Detection of a genotype-virulence
association with the Ghanaian MSV isolates might there-
fore require an analysis of infections initiated from cloned
viral genomes.
The current predominance of MSV-A1 in Ghana is a
mixed blessing. Although MSV-A1 displays a higher
degree of pathogenicity than the other known MSV sub-
types [9], the apparent genetic uniformity of the Ghanaian
MSV-A population may mean that MSV-resistant maize
genotypes that are suitable for growth in Ghana need only
fare well against MSV-A1 variants. It is noteworthy in this
regard that of the 79 infected maize fields surveyed, 15
were planted with ‘‘certified’’ MSV-resistant maize geno-
types (average incidence = 13.733 % and average MSD
severity = 2.754). The presence of MSD in these fields
strongly suggests that at least some of the MSV-A1 variants
that we have characterized here (and perhaps all) already
had the capacity to infect many West African MSV-resis-
tant maize genotypes. These 51 cloned isolates will
therefore be a particularly valuable resource in Ghana
during the development of the next generation of MSV-
resistant maize genotypes.
Acknowledgments The molecular work described in this manu-
script was supported by grants from the National Research Founda-
tion of South Africa awarded to DPM and AV. We are also grateful to
the Alliance for Green Revolution in Africa (AGRA) through the
West Africa Centre for Crop Improvement (WACCI), University of
Ghana, Legon, for their financial support for field work awarded to
AO.
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