Molecular detection of two cassava Begomoviruses in some parts of Southern Nigeria

Cassava mosaic disease (CMD), caused by an array of is the most economically important viral disease of cassava in sub-Saharan Africa. The most frequently reported in West Africa are African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV). In this study, 42 cassava leaves and 30 symptomatic weeds belonging to the Asteraceae, Cucurbitaceae and Leguminosae families were collected from backyard gardens in Edo, Ondo, Anambra, and Delta States in 2009. Deoxyribonucleic acid (DNA) extracts from these leaves were tested for ACMV and EACMCV in a multiplex polymerase chain reaction (PCR) assay. The PCR primers used were designed to amplify the replicase regions of DNA-A components of both viruses. Most of the cassava plants within the survey area were either symptomless or showed mild symptoms. ACMV was detected in 16% of cassava leaves from Edo State but not in any of the cassava leaves from the other three states. One weed sample each from Edo State (5.56%) and Ondo State (10%) were also positive for ACMV. EACMCV was not detected in any of the samples tested. The low virus occurrence observed from PCR results and the observed low incidence of the CMD characteristic mosaic symptoms on cassava leaves in the states sampled may be attributed to the use of CMD resistant or tolerant cassava varieties, and may be a result of the massive distribution of virus resistant cassava cuttings to these States by the International Institute of Tropical Agriculture (IITA).


INTRODUCTION
Cassava (Manihot esculenta) is one of the leading food and feed plants of the world, particularly in Africa (FAO, 2008;Nassar and Ortiz, 2007).It is grown mainly for its enlarged starch-filled root, which is used for human consumption and, commercially, for the production of animal feed and starch-based products (O' Hair, 1990).Young tender cassava leaves are consumed as vegetables in many regions in Africa as a source of protein, vitamins and mineral salts (Legg and Fauquet, 2004).
Cassava mosaic disease (CMD) is the major constraint to cassava production in Africa and is caused by eight distinct of the family Geminiviridae, commonly referred to as Cassava mosaic (CMGs) (Fauquet and Stanley, 2003;Thresh and Cooter, 2005;Fauquet et al., 2008).CMD results in stunting and severe reduction in the yield of the desired tuberous cassava root and is thus a production threat to cassava which feeds over 200 million people in sub-Saharan Africa.CMD has been reported as occurring at varying levels of incidence throughout the cassava belt of Africa (Ogbe et al., 2003b;Thresh et al., 1997).For several years after African cassava mosaic virus (ACMV) *Corresponding author.E-mail: angelaeni@gmail.com.Tel: +234 086 393 9991.
was confirmed as the causal agent of CMD, it remained the only known causal agent of CMD in Nigeria until the 1990s when East African cassava mosaic Cameroon virus (EACMCV) and several variants of the EACMV were diagnosed as additional causative agents (Ogbe et al., 2003a;2003b;Ariyo et al., 2005;Ogbe et al., 2006;Alabi et al., 2008b).Cassava plants infected with CMGs express a range of symptoms depending on factors like the virus strain or species, environmental conditions and the susceptibility of the cassava host (Legg and Thresh, 2003).CMDaffected plants are stunted with conspicuous foliar symptoms such as mosaic and chlorosis interfering with the photosynthetic ability of the plant to produce food for storage in the roots, thus, infected plants produces greatly diminished or no tuberous roots.
This study reports the testing of cassava and symptomatic weeds collected from backyard gardens in some states in Southern Nigeria, for ACMV and EACMCV.

MATERIALS AND METHODS
Sample leaves from cassava plants showing the CMD characteristic mosaic symptom were collected from farms in Edo, Delta, Ondo and Anambra States in July, 2009.A few non-symptomatic cassava leaves were also collected.Leave samples were also collected from symptomatic Chromolaena odorata (L.) King and Robinson, Centrosema pubescens Benth., Senna alata (Linn.)Roxb., and Cucurbita spp weeds within the vicinities of the sampled gardens, to ascertain their role as alternate/reservoir hosts for the viruses.Leaf samples were kept on ice while in the field and processed the same day by splicing and drying (rapidly) in anhydrous calcium chloride.Total deoxyribonucleic acid (DNA) was extracted from cassava and weed samples as previously described by Dellaporta et al. (1983).
The PCR amplified products were resolved by agarose gel electrophoresis and visualized under ultraviolet (UV) light using a gel documentation system.A 100 bp DNA molecular weight marker (Promega Corporation, Madison, W1) was run in each gel as a reference to estimate the size of the virus-specific DNA band in the PCR amplified products.

RESULTS
A total of 42 cassava leaves and 30 weeds were collected for virus testing.Seven (7) of the cassava leaves were non-symptomatic, while 35 were symptomatic.All the weeds tested showed leaf mosaic Eni and Fasasi 1351 symptoms, and they were tested to determine their role as alternative host for the viruses.The distribution of leaf mosaic both on cassava and weeds across the states is shown in Figure 1.The expected amplicon sizes for ACMV and EACMCV are 368 and 650 bp, respectively.Only 8.33% of the 72 samples produced the expected amplicon size of 368 bp for ACMV.Four of the positive samples were cassava leaves from Edo state; three of these were symptomatic, while one was non-symptomatic (Table 1

DISCUSSION
This study describes our quest to determine the occurrence of ACMV and EACMCV in cassava and associated weeds in backyard gardens in some parts of Southern Nigeria.Most cassava plants in the gardens surveyed were non-symptomatic.The high incidence of uninfected cassava plants observed in this study differs from reports from previous surveys in Nigeria where high incidence of CMD symptoms were reported in cassava fields (Ogbe et al., 2003a(Ogbe et al., , 2003b(Ogbe et al., , 2006)).This difference may be due to the massive distribution and use of healthy, resistant, and/or tolerant cassava varieties by the farmers in the sampled areas (Manyong et al., 2000).This clearly indicates that increasing farmers' awareness on the cause and nature of a disease and possible control measures ensures their participation in the eradication of the diseases.The control of CMD is achieved mainly by the use of resistant/tolerant varieties and by employing adequate phytosanitry techniques of destroying diseased plants and by the use of healthy stems.
The positive detection of ACMV in some of the weeds tested confirms previous reports that weeds serve as alternate host for (Monde et al., 2010;Alabi et al., 2008a;Ogbe et al., 2006) and further buttresses the role of phytosanitation in the control of ACMV.This information is also of epidemiological importance because infected weeds may serve as virus reservoirs and inoculum foci.
Although, most positive samples were symptomatic, the detection of ACMV in one of the non-symptomatic cassava leaves indicates the possibility of latent infection.This phenomenon had been previously reported during field surveys in 2002 and 2003 (Ogbe et al., 2006), and further highlights the need for laboratory certification of cassava planting materials.
The negative EACMCV result obtained for all the leaves tested may indicate either the absence of the virus in the fields sampled, or an extremely low incidence.The high incidence of negative symptomatic cassava leaves  may be indicative of the occurrence of other not tested for in this study and requires further investigation.CMD remains a threat to cassava production in several African countries.The chances of emergence of new strains/variants of the causative are also very high due to the frequent reports of dual/multiple infections (Fondong et al., 2000;Harrison et al., 1997).Constant surveillance, phytosanitation, and improvement on existing diagnostics will continue to play major roles in ensuring that Nigeria retains its non-epidemic status and low epidemic index (Legg and Owor, 2003).It is also important to ensure that existing quarantine measures continue to be enforced.

Figure 1 .
Figure 1.Distribution of leaf mosaic symptom on cassava leaves and weeds collected from Edo, Ondo, Anambra, and Delta States, Nigeria.

Table 1 .
Summary of cassava and weed samples collected and the result of multiplex PCR testing for ACMV and EACMCV in the surveyed samples from Edo, Delta, Ondo and Anambra states, Nigeria.
).One C. pubescens leaf sample from Edo State and one S. alata sample from Ondo State tested positive for ACMV.None of the samples from Delta and Anambra States tested positive to ACMV, and EACMCV was not detected in any of the samples tested in all the States.