White yam Dioscorea rotundata is a popular starchy staple in Ghana.
One of the major constraints to yam production in general is the effect of pest
and diseases including those caused by viruses (Anonymous,
1992). Viruses adversely affect Dioscorea species by reducing vigour
and subsequently the yield of the tuber (Amusa et al.,
2003; Mandal, 1993; Coursey, 1967).
An estimated yield reduction of 50% was reported by Amusa
et al. (2003) as a result of YMV infection on D. rotundata
Viruses that infect yams throughout the world belong to five genera, namely,
potyvirus, potexvirus, badnavirus, carlavirus and cucumovirus (Brunt
et al., 1996). In West Africa, however, viruses from the genera,
potyvirus is the most widespread (Thouvenel and Fauquet,
1979). Viruses in the other genera have also been detected in the various
locations and countries from different yam species.
Symptoms of infection are several; notable among them being the leaf mosaic.
Other symptoms include leaf distortion, leaf malformation, chlorosis and in
severe cases, shoe-stringing of the vines (Rossel and Thottappily,
1985). Some infections may also be symptom-less or latent.
In Ghana Yam mosaic virus (YMV), a potyvirus is reported to be the most
commonly detected virus on D. rotundata (Olatunde and
Hughes, 1999). Thus, in almost all cases, white yam virus disease is attributed
to YMV alone. However, in the yam belt of West Africa, outside Ghana, other
viruses belonging to the genus badnavirus, cucumovirus and other potyviruses
have been isolated (Rechaus and Nienhaus, 1981; Hughes,
1986; Fauquet and Thouvenel, 1987). It is possible
that some of these viruses may also be present in Ghana due mainly to the exchange
of yam planting materials across the sub-region and vector activity.
A study was thus carried out aimed at detecting the existence of any of the known yam viruses other than YMV in Ghana. The findings of this study would be very useful in the management of viral diseases of yam especially the use of virus-free planting materials which will improve yield thus enhancing livelihoods.
MATERIALS AND METHODS
In a survey, leaves of D. rotundata showing virus-like symptoms were collected from ten important yam growing districts in Ashanti and Brong Ahafo regions, situated in the forest and forest-transition zones of Ghana. Sampling was done in farms situated at Fumesua and Tanoso (Kumasi district); Ejura and Sekyeduamasi (Ejura-Sekyedumasi district); Mampong (Mampong district) all in the Ashanti region of Ghana. Duayaw Nkwanta and Susanho (Tano North district), Sunyani, Fiapre, Chira and Heman (Sunyani district); Kintampo, Jema, Barbatorkuma and Beposo (Kintampo district), Techiman, Aworowa, Tanoso and Nipahiamoa (Techiman district); Wenchi, Ayayo, Droboso and Buoko (Wenchi district); Nkoranza, (Nkoranza district) and Atebubu, Prang and Amantin (Atebubu district) all in the Brong Ahafo region. In each town 3-4 farms were visited. Leaf samples were collected in the months of June/July, 2004 when plants were about four months old. Eight to ten plants were sampled from each site visited and leaves showing viral symptoms collected. They were labeled and then kept in an ice chest at a temperature of about 4°C and then sent to the laboratory for sero-diagnosis.
The enzyme linked immunosorbent assay (ELISA) technique was used to virus index the samples. Antibodies homologous to Yam mosaic virus (YMV), Cucumber mosaic virus (CMV), Dioscorea alata potyvirus (DAV), genus Potyvirus and Dioscorea alata badnavirus (DaV), genus Badnavirus, kindly supplied by the virology laboratory of the International Institute of Tropical Agriculture (IITA) were used.
To detect Dioscorea alata potyvirus, genus Potyvirus, Dioscorea
alata badnavirus, genus, Badnavirus and Cucumber mosaic virus,
genus Cucumivirus, the protocol followed was by developed by Edwards
and Cooper (1985) and modified by Hughes and Thomas
(1988). Three replications of each sample were used for the detection.
ELISA microtitre plates (NUNC) were coated with (100 μL) Protein A solution in coating buffer and incubated for 2 h at 37°C after which ground samples (1 g/2 mL of extraction buffer), were added and incubated overnight at 4°C. The detecting antibody was added and incubated for 2 h at 37°C after which Protein-A alkaline phosphatase conjugate diluted in conjugated buffer (1:50,000) was added and incubated for same time period and temperature. Between each step of ELISA procedure (incubation period) the plates were washed thrice at 3 min intervals. 0.01 g/10 mL of p-nitrophenyl phosphate salt in substrate buffer was finally added and incubated overnight at room temperature for one hour.
To detect YMV in our samples, the triple antibody sandwich ELISA (TAS ELISA)
procedure was followed.
This was as described by Adams and Barbara (1982),
Barbara and Clark (1982). ELISA plates were coated with
100 μL YMV polyclonal antibody diluted in coating buffer (1:1000) and incubated
for 2 h at 37°C after which samples (1 g/2 mL extraction buffer) were added
to the wells and incubated overnight at 4°C.
YMV monoclonal antibody (1:1000 in PBS-Tween 20) was then added to wells and incubated for 2 h at 37°C. Goat anti-mouse alkaline phosphatase conjugate diluted in conjugate buffer was added to wells and incubated for 2 h at 37°C. Between each step of ELISA procedure the plates were washed thrice at 3 min intervals. 0.01 g/10 mL of p-nitrophenyl phosphate salt in substrate buffer was finally added and incubated at room temperature and overnight.
For both PAS and TAS ELISA Protocols, Optical Density (OD) values of contents
in the wells were determined by using microplate reader at 405 nm wavelength.
A sample was considered positive when the OD value was at least twice that of
healthy control (Sutula et al., 1986).
One hundred and sixty leaf samples were analysed. None of the antisera raised against DaV and CMVreacted positively to antigens in yam samples (Table 1).
However antiserum raised against YMV reacted positively to antigens in 68 (38%) samples, while that raised against DAV reacted positively to 36 (20%) samples. Antiserum raised against YMV and DAV which occurred in a mixture reacted positively to antigens in 20 (11%) samples. In all 72 (41%) samples reacted negatively with antisera raised against YMV, DAV, DaV and CMV even though they showed virus-like symptoms.
The Wenchi district recorded the highest incidence of samples responding to antibody homologous to YMV 75%, followed by the Kumasi district 65%, Kintampo district 65%, Sunyani 40% Ejura/Mampong districts 36.8%, Atebubu/Prang districts 31.5%, Techiman district 20% and Tano North district 10.5% in that order while all the samples collected in the Nkoranza district tested negative
For the Dioscorea alata potyvirus, DAV, Techiman district recorded the highest incidence of samples testing positive 80% followed by Wenchi district 60%, Kintampo 20% and Sunyani 20%. Samples from the remaining districts tested negative.
The results as shown in Table 1 clearly indicate the widespread nature of YMV in the study areas. This is followed by Dioscorea alata potyvirus (DAV), also known as yam virus 1), which is more prevalent around Wenchi, Techiman, Sunyani and Kintampo districts. Cucumber mosaic virus and Dioscorea alata badnavirus, were not detected in any of the samples tested, possibly indicating their absence in the study areas.
|| Viruses detected with ELISA on D. rotundata leaf samples
from yam growing districts in Ghana
According to Thouvenel and Fauquet (1977), Brunt et al.
(1996), YMV is the most dominant virus on white yam throughout West Africa.
It is not surprising that it was the dominant virus found in most of the samples
Dioscorea alata potyvirus is also common particularly on Dioscorea
alata, it was first detected in Togo, Rechaus (1986). Hughes
(1986) also detected the virus on D. alata from the Caribbean and
Indonesia. Odu (1997) detected the virus on D. alata
from Nigeria. Previous findings indicate that DAV has never been isolated
or detected on D. rotundata in Ghana (Olatunde and
Hughes, 1999). The detection of DAV on white yam as has been shown in our
results thus contradicts Olatunde and Hughes (1999) who
reported that DAV was limited to D. alata in Ghana.
In some of the samples YMV and DAV were also found to coexist. This result
is again the first report of such infection in Ghana. This finding also contracts
Olatunde and Hughes (1999) who reported that YMV infection
on white yam never coexisted with DAV. In their report on a comprehensive virus
survey carried out in farmers fields in seven regions of Ghana in 1998
and 1999; two viruses; YMV and DAV were both detected on Dioscorea species
but not in mixed infections. While YMV was detected on D. rotundata and
D. alata cultivars, DAV was detected only in D. alata (Hughes
and Olatunde, 1999).
Generally it is not surprising that DAV has transferred from D. alata to D. rotundata. This is because both species belong to the same genus; Dioscoreacae. Again DAV and YMV are different strains of the virus genus belonging to the potyvirus group.
Forty one percent of the samples collected responded negatively to all the
antibodies. This could de due to the fact that the virus concentration in them
might have been low, probably because of the time of collection. Also, varietal
characteristics or strain of virus might have been different, or due to the
presence of other viruses, (Van Regenmortel, 1982; Matthews,
Improvement in the yield of white yams cannot be achieved if viral diseases continue to be prevalent. The most effective way of controlling plant viral diseases is to breed for crop cultivars that are resistant/tolerant to virus infections. The detection of DAV on white yam should signal to yam virus disease researchers and breeders the need to develop comprehensive virus cleaning measures to clean white yam and indeed all yam planting materials from all known yam viruses to realize production of completely virus-free planting materials for better yields to be achieved.
We are very grateful to the Root and Tuber Improvement Programme (RTIP) and the Agricultural Sub-Sector Improvement Programme for their financial support. We are also thankful to International Institute for Tropical Agriculture (IITA), Nigeria.