Plant
2016; 4(6): 108-113
http://www.sciencepublishinggroup.com/j/plant
doi: 10.11648/j.plant.20160406.18
ISSN: 2331-0669 (Print); ISSN: 2331-0677 (Online)
Occurrence and Distribution of Begomoviruses Infecting
Cassava in Western Kenya
Mariam Nyongesa Were1, Benard Mukoye1, *, Aggrey Keya Osogo2, Bonphace Collins Mangeni1,
Paul Ateng’a Nyamwamu1, Vitalis Kalor Ogemah1, John Vincent Muoma1, Stephan Winter3,
Hassan Karakacha Were1
1
Department of Biological Sciences, Masinde Muliro University of Science and Technology, Kakamega, Kenya
2
Department of Biological Sciences, Kibabii University, Bungoma, Kenya
3
Leibniz-Institute DSMZ Plant, Virus Department, Braunschweig, Germany
Email address:
Claris.nyongesa@yahoo.com (M. N. Were), btemukoye@gmail.com (B. Mukoye), aosogokeya@yahoo.co.uk (A. K. Osogo),
barasamangeni@gmail.com (B. C. Mangeni), pnyamwamuta2@yahoo.com (P. A. Nyamwamu), vogemah@mmust.ac.ke (V. K. Ogemah),
jmuoma@gmail.com (J. V. Muoma), Stephan.winter@dsmz.de (S. Winter), werehkde@yahoo.com (H. K. Were)
*
Corresponding author
To cite this article:
Mariam Nyongesa Were, Benard Mukoye, Aggrey Keya Osogo, Bonphace Collins Mangeni, Paul Ateng’a Nyamwamu, Vitalis Kalor
Ogemah, John Vincent Muoma, Stephan Winter, Hassan Karakacha Were. Occurrence and Distribution of Begomoviruses Infecting Cassava
in Western Kenya. Plant. Vol. 4, No. 6, 2016, pp. 108-113. doi: 10.11648/j.plant.20160406.18
Received: September 29, 2016; Accepted: October 11, 2016; Published: November 7, 2016
Abstract: Cassava (Manihot esculenta Cranz) is an important food staple in Busia, Homabay, Siaya, Migori, Kwale, Kilifi
and Marakwet Counties and is a secondary food crop for many Kenyans. The current yields of 3-4 tons/ha obtained in Western
Kenya are far below world averages and this is largely attributed to pests and diseases. The usual practice of retaining some
seed cuttings from the current ware crop or buying them from neighbours, leads to accumulation of viral diseases most
important of which are caused by begomovirus infections. A survey for cassava mosaic disease (CMD) was carried out in main
cassava growing areas of Western Kenya with a view to determine incidence and distribution of the causal viruses. A total of
33 cassava farms in seven sub-counties in Western Kenya were covered. Leaf samples were collected and analysed
serologically and by molecular means. Cassava plants in most farms were severely affected by cassava mosaic disease. Disease
incidence in farms ranged between 2% to 54%. Three cassava infecting begomoviruses, African Cassava Mosaic Virus
(ACMV), East African Cassava Mosaic Virus (EACMV) and East African Cassava Mosaic Virus – Ugandan variant (EACMVUg) were found in the collected samples, with EACMV-Ug being most prevalent followed by EACMV. These are interesting
findings given that in the past surveys, ACMV was the most abundant virus in the area. To increase cassava yields, it is
recommended that cassava farmers be educated on cassava diseases and their control.
Keywords: Begomoviruses, Western Kenya, Manihot Esculenta
1. Introduction
Cassava is an important staple food crop in Busia,
Homabay, Siaya, Migori, Kwale, Kilifi and Marakwet
Counties of Kenya and is a secondary food crop for many
Kenyans whose staple diet is based on cereals, particularly
maize [1]. Previous work [2] established that sweet-potato
and cassava are important food security crops when maize,
the major staple, is in short supply or in years of drought.
In urban areas there is a growing demand for good quality
fresh cassava roots and for processed products such as flour
and chips. Cassava is usually grown in rain fed, low-input
systems and the yields obtained in Kenya of 11 t/ha [3] are
among the poorest in the world compared to 20 t/ha in
Nigeria and about 24t/ha on experimental stations. Cassava is
propagated vegetatively through planting stem cuttings.
Although there are many constraints to production, every
analysis has shown that the single most important constraint
in the whole country is the lack of high quality disease-free
planting material in the form of stem cuttings. A second
Plant 2016; 4(6): 108-113
important constraint is the lack of grower awareness and
knowledge of pests and diseases or how to manage them. The
usual practice of farmers to retain stem cuttings from the
current crop being produced for consumption or buy them
from a neighbour leads to high incidence of whitefly
transmitted virus diseases namely, Cassava mosaic disease
(CMD) and Cassava brown streak disease (CBSD). The
causal viruses of CMD are: African cassava mosaic virusKenya (ACMV-K), and East African cassava mosaic virus
(EACMV), East African cassava mosaic Zanzibar virus
(EACMZV), Uganda variant strain of the EACMV
(EACMV-Ug) and East African cassava mosaic Kenya Virus
(EACMKV) while Cassava brown streak virus (CBSV) and
Cassava brown streak Uganda virus (UCBSV) cause CBSD
[4 -7]. These viruses are spread by whiteflies rapidly during
the growing season or by infected cuttings in subsequent
seasons. Incidences of up to 100% have been found in Kenya
[8]. Moreover, if crops are grown in land contaminated by
bacteria or nematodes the cuttings become infected leading to
bacterial or nematode infection in subsequent generations.
Surveys done previously [4, 6, 8, 9] found ACMV,
EACMV, EACMKV and EACMV–Ug as the most prevalent
viruses in Western Kenya but no recent survey in the region
to determine the status of CMD causal viruses has been done.
This paper reports the results of a survey undertaken in the
main cassava-growing regions of Western Kenya to monitor
the dynamics of the relative prevalence of CMD
begomoviruses.
2. Materials and Methods
2.1. CMD Survey
A diagnostic survey for CMD was conducted in Bungoma
South, Busia, Homabay, Rachuonyo, Rongo, Teso South and
Ugenya sub-counties of Western Kenya. Farms were
surveyed during the long rains of May/June 2011 to take
advantage of the vigorous vegetative growth of cassava at the
time. Cassava fields were selected at regular intervals of
between three and five kilometres along the main roads and
occasionally feeder roads. The number of fields sampled per
county depended on the availability of cassava farms at the
time of survey. Sampling was done on plants along diagonals
and the number of samples collected per field depended on
the variability of symptoms and filed size. Disease incidence
was calculated as a percentage of symptomatic plants to the
total number of plants along the sampling diagonals.
Coordinates were taken at each sampling site using a global
positioning system (GPS) device (Magellan Triton ‘Windows
CE Core 5.0 X11-15302).
Visual symptoms were assessed as described earlier [8]
and classed as mosaic, dwarfing, chlorosis, or a combination
of these.
Data obtained from the surveys (altitude, and virus disease
incidence) were recorded in Microsoft Excel spreadsheets,
while correlation and regression analysis was done using the
computer software Statistical Package for Social Scientists
109
(SPSS).
2.2. Virus Detection by ELISA
Leafy samples from 3 to 5 cassava plants per farm were
collected and tested using Triple antibody sandwich enzyme
linked immunosorbent assay (TAS-ELISA) as described
previously [10] using polyclonal antibodies raised against
CMD viruses for coating and monoclonal antibodies specific
to ACMV and EACMV viruses for detection. Microtiter
plates (Greiner Microlon medium binding) and reagents for
the virus tests were obtained from the German collection of
Microorganisms and Cell cultures (DSMZ), Braunschweig,
Germany. Tests were done following the manufacturer’s
instructions. Each ELISA plate contained positive and
negative control samples and samples were tested in single
wells. Absorbance values were recorded at 405 nm (A405)
using
a
Titertek
Multiscan®
MCC/340
model
spectrophotometer (Labsystems Co., Finland).
Samples whose absorbance values (A405) exceeded the
negative control values by a factor of two were considered
positive. Some plates were scored visually and samples
considered positive if the colour of substrate in the sample
wells was clearly stronger than in the negative control wells.
2.3. PCR-Mediated Amplification of Cassava Begomovirus
Species DNA-A Fragments
Total DNA was extracted from young virus-infected
cassava leaves essentially as described by [11]. Amplification
of viral sequences was done using degenerate universal
geminivirus primers [12]. For detection and differentiation of
cassava begomoviruses, ACMV, EACMV-Ug, sequences in
single and in mixed virus infections, a polymerase chain
reaction (PCR) approach was followed. PCR tests were
conducted using primers specific for ACMV, EACMV and
EACMV-Ug. For ACMV, (ACMV AL1/F, ACMV ARO/R
[4], EACMV (EACMVA391/ EACMVA2469) and EACMVUg (UV AL1/F1/ ACMV CP/R3) [3] primer combinations
were used. Amplification by PCR proceeded as follows:
Total plant DNA (1 µl) was subjected to PCR in a total
reaction volume of 50 µl. The reaction consisted of 2.5 µl
MgCl2, 1 µl of 20 pmol of each primer, 5 µl of 10x Taq
polymerase buffer, 1 µl 5 mM dNTPs, and 0.5 µl or 2.5 units
of Taq DNA polymerase (Toyobo, Osaka, Japan). The
cycling profile was 3 min of denaturation at 94°C followed
by 35 cycles of denaturation, annealing and DNA extension
of 1 min at 94°C, 1.5 min at 52°C, and 1.5 min at 72°C
respectively. A final extension period of 10 min at 72°C
finished the PCR amplification.
3. Results
The surveyed farms were located between longitudes
00190850E (a farm in Ugenya) and 99553780E (a farm in
Rachuonyo) and latitudes 06272000N (a farm in Busia) and
0913420N (a farm in Rachuonyo). The lowest altitude of the
surveyed farms was 1181m above sea level (m asl) (a farm in
110
Mariam Nyongesa Were et al.: Occurrence and Distribution of Begomoviruses Infecting Cassava in Western Kenya
Teso South) while the highest was 1496 m.asl (a farm in
Rongo).
3.2. TAS-ELISA
Of the 53 samples tested, 44 were positive for cassava
begomoviruses. An interesting feature was that although most
samples collected from all sub-counties had cassava
begomoviruses, a few samples from Homabay (3 out of 7),
Teso (4 out of 7) and Ugenya (2 out of 8) were negative. This
was despite the fact that most samples collected had typical
CMD symptoms. Furthermore, because ELISA could not
differentiate between the begomoviruses, a PCR test was
conducted.
3.1. CMD Symptoms and Incidence
Symptoms observed in the field on cassava infected with
CMD ranged from chlorotic spots, leaf curling, bright mosaic
and stunting of severely affected plants.
Cassava mosaic disease was present in all the sub-counties
surveyed. The highest CMD incidence (54%) was observed
in a farm in Bungoma South while a farm in Rongo had the
lowest (2%). The mean disease incidence was highest in Teso
South (30%) while Rongo had the lowest (11%). Overall, the
mean incidence for all the sub-counties surveyed was 19%.
(Fig. 1)
3.3. PCR
Results of the PCR tests conducted using primers specific
to ACMV, EACMV and EACMV-Ug detected ACMV,
EACMV and EACMV-Ug in samples collected from all
surveyed areas except Rongo where there was no EACMVUg (Table 1 and Fig.2). Mixed infections with all the three
viruses (ACMV, EACMV and EACMV-Ug) were found in 9
of the 30 samples tested, ACMV and EACMV were in 7
samples while EACMV and EACMV-Ug, were in 9 samples.
Two samples (from Rongo and Rachuonyo) had single
EACMV infections while a sample from Bungoma South had
a single infection with ACMV. It is interesting to note that
EACMV-Ug was never found infecting cassava alone but
was always in mixed infections either with the other two
combined or with EACMV. EACMV was the most dominant
virus (found in 26) of the 30 samples) followed by EACMVUg found in 18 samples while ACMV occurred least in 17
samples. Surprisingly, asymptomatic cassava sample
obtained from Ugenya as a healthy control, tested positive by
ELISA and was also found to have mixed infection of
ACMV, EACMV and EACMV-Ug by PCR analysis.
Figure 1. A bar graph showing mean CMD incidence per Sub-county.
Legend: bungos-Bungoma south, tesos- Teso South, busia – Busia, ugen –
Ugenya, rach, Rachuonyo, homa- Homabay, rongo, Rongo
Table 1. TAS-ELISA and PCR results obtained from analysis of cassava samples for cassava begomoviruses.
Sub-county
Bungoma S.
Busia
Homabay
Rachuonyo
Rongo
Teso S.
Ugenya
ELISA
+ve
17
5
4
7
2
3
6
-ve
0
0
3
0
0
4
2
PCR +ve
# Tested
7
2
4
6
1
6
4
A
1
0
0
0
0
0
0
E
0
0
0
1
1
0
0
Legend: A=ACMV, E=EACMV, U=EACMV-Ug
a
U
0
0
0
0
0
0
0
A+E
1
0
2
2
0
1
1
A+U
0
0
0
0
0
0
0
E+U
2
0
2
2
0
2
1
A+E+U
3
2
0
1
0
2
1
Plant 2016; 4(6): 108-113
111
b
Figure 2. a. Gel electrophoresis of PCR amplification of cassava (ACMV& EACMV-Ug) begomovirus DNA (expected band size: ACMV=975 bp, EACMVUg=). Lanes 1 - 3; leaf samples from Bungoma South (11b, 12a and 12b), Lanes 4 and 5; Ugenya (13a and 13b), Lanes 6 and 7; Homabay (14a and 14b),
Lane 8; Busia (15) and Lanes 9 – 10; Rachuonyo (16a and 16b). b. Gel electrophoresis of PCR amplification of cassava (EACMV) (expected band size
EACMV=520 bp). Lanes 1 and 2; leaf samples from Bungoma South (12a and 12b), Lanes 3 and 4; Ugenya (13a and 13 b), Lanes 5 and 6; Homabay (14a
and 14b), Lane 7; Busia (15), Lanes 8 – 10 and 13; Rachuonyo (16a, 16b, 16c and 18a), Lanes 11 and 12; Teso South (17a and 17b).
3.4. Distribution of ACMV, EACMV and EACMV-Ug in
Western Kenya
Based on ELISA and PCR results, a distribution map of
cassava begomoviruses occurring in Western Kenya was
drawn (Fig.3). EACMV was the dominant virus found
occurring in all surveyed areas followed by EACMV-Ug
which, in all cases, was found in mixed infections. The other
virus ACMV was only found alone in a sample from
Bungoma South and was the least occurring virus in the
region.
Figure 3. Map of Western Kenya (not drawn to scale) showing the
distribution of ACMV, EACMV and EACMV-Ug in the surveyed areas (In
red font).
4. Discussion
The highest CMD incidence was found to occur at levels
of up to 54%. Such levels are considerably high and continue
to increase with successive croppings. Although [13]
reported successful restoration of cassava production in
Western Kenya after introduction of new varieties with
resistance to CMD this study found some of these varieties
with high CMD incidence. This could mean the varieties
either had some resistance which had broken down or that
they are susceptible to the Western Kenya strains of the
begomoviruses. The other scenario could be that since
material was new, it was clean and therefore needed time to
get infected in the field. With the current practice of farmers
planting cuttings immediately after harvest, an epidemic
could erupt similar to that of the 1990s if nothing is done in
the near future. Provision of clean planting material to
farmers seemed to lower the spread of the disease an
observation supported by [7] who observed that due to lack
of sustainability in providing clean material arrangements,
there is a conspicuous resurgence of the viral diseases again.
The CMD incidence was not significantly different among
the surveyed districts. This implies that farmers in these areas
get planting material from the same sources and cultural
practices are similar. According to [14] and [15], variability
in the pathological incidences of CMD is often attributed to
several factors such as different strains of virus, susceptibility
and age of the host plant and environmental factors such as
solar radiation and temperature. Therefore, similar incidences
witnessed in this study may be attributed to the fact that the
surveyed sub-counties, though in different ecological zones,
they had no significant difference in the altitudes.
Furthermore, in addition to the weather conditions being the
same, there was no significant difference in age of cassava
crops. A striking feature of this study was that there was no
farm with 0% CMD incidence, an indication that all cassava
varieties in the surveyed area were susceptible to the viruses
and that all farms were infected. Surprisingly, CMD was
known to all farmers who were covered in the survey, but no
single farmer looked keen to contain or try to do so by
phytosanitary means. This is supported by the fact that most
farmers used and recycled their own cuttings saved from a
previous crop as source of planting material leading to
propagation of the viruses if the parent material was infected.
This confirms the findings by [15] that CMD appears to
originate mostly from unhealthy cassava cuttings used for
planting.
Several CMD causal virus species ACMV and EACMV
and an EACMV variant EACMV-Ug, were detected by TAS-
112
Mariam Nyongesa Were et al.: Occurrence and Distribution of Begomoviruses Infecting Cassava in Western Kenya
ELISA and by PCR. Although TAS-ELISA was cheap and
relatively easy to carry out, it was not possible to differentiate
the viruses using the method due to unavailability of
monoclonal antibodies for EACMV-Ug and EACMKV.
Moreover, viruses in mixed infections could not be
differentiated hence the need for molecular diagnosis.
However, with appropriate and high quality antisera, this
method can be very useful in places where PCR is either not
available or just expensive (reagents, equipment and
expertise) to sustain. On the other hand, although the primers
used for PCR were robust and very precise, they could not
detect EACMKV or EACMZV. New additional primers need
to be derived that can detect these viruses.
From PCR results, 83% of the positive cassava samples
were infected with EACMV, 60%, with EACMV-Ug and
57%, ACMV. This makes EACMV and EACMV-Ug the
most abundant viruses in Western Kenya. Before EACMVUg was reported, Western Kenya was predominantly an
ACMV zone with EACMV being geographically located
along the coast or the Eastern side of the Rift valley. A few
reports [8] had found EACMV in Kisii and Alupe, since then
there has been no comprehensive surveys for the viruses in
the area. It was reported then that EACMV-Ug was very
virulent [4] and that it was displacing ACMV from ACMV
traditional areas. This study has found ACMV to be the least
predominant virus and confirms earlier workers [8] fears.
However, it is not clear how ACMV has been displaced by
EACMV and EACMV-Ug.
Some samples collected from variety TME 14 with no
symptoms were found to have mixed infections of ACMV,
EACMV and EACMV-Ug after PCR tests. This clearly
shows that the viruses can be latent in the plant without
symptoms manifestation. Such varieties may be tolerant to
the viruses and planting them could be dangerous as this
could act as a source of infection and could fuel the spread of
the disease to more susceptible varieties.
With regard to distribution of begomoviruses in Western
Kenya, the three begomoviruses (ACMV, EACMV and
EACMV-Ug) were found to be widely distributed in the
surveyed area. In all cases, mixed infections of these three
viruses were detected in samples from most farms. EACMV
was the most predominant virus followed by the strain
EACMV-Ug. This slightly differs with earlier findings [1618] that none of disease surveys conducted in Sub-Saharan
Africa have reported the incidence of the EACMV virus
suggesting that this ‘wild-type’ EACMV has been overtaken
in nature by the more fit recombinant strains. Furthermore,
since EACMV-Ug was first reported in Uganda in late 1980s,
it has spread to affect nine countries in East and Central
Africa [19, 20]). The reasons for this difference could be that
none of the above surveys was done in Kenya, and since
EACMV has never been reported in Uganda, it is not
surprising that the findings confirm this view. In Western
Kenya, all the three viruses were widespread in the entire
surveyed area but EACMV-Ug has displaced most of the
ACMV in most farms in Western Kenya as earlier predicted.
[8].
5. Conclusions
CMD incidence is very high in Western Kenya, which
requires urgent attention.
PCR is the most reliable and sensitive means of
differentiating the begomoviruses, however, in places where
the tests may not be possible by this means, TAS-ELISA may
suffice if new MAbs are produced in line with the changing
virus dynamics in the field.
All the three cassava begomoviruses (EACMV, EACMVUg and ACMV) are present in Western Kenya mainly in
mixed infections. EACMV and EACMV-Ug have displaced
ACMV from most of the areas that were traditionally
occupied by the latter.
Farmers use own planting materials or those from
neighbours most of which are not clean and therefore serve
as source of virus.
For effective control of CMD, farmers need to be
sensitized on the benefits of using clean planting material,
which should be made available.
Acknowledgments
This study was funded by the National Commission for
Science, Technology and Innovation (NACOSTI), Kenya
(Ref. NCST/ST&I/4th Call M.Sc/095).
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