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1 Department of Disease and Stress Biology, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
2 National Crops Resources Research Institute, Namulonge, PO Box 7084, Kampala, Uganda
Correspondence
Rob W. Briddon
rob.briddon{at}gmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Plant Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, PO Box 577, Faisalabad, Pakistan. 
Present address: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
A supplementary table showing the primers used in this study is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Cassava mosaic disease (CMD), the major constraint to cassava production in Africa, is caused by a number of distinct begomoviruses (family Geminiviridae) (Thresh & Cooter, 2005). To date, seven begomovirus species have been identified in association with CMD, namely African cassava mosaic virus, East African cassava mosaic virus, East African cassava mosaic Cameroon virus, East African cassava mosaic Kenya virus, East African cassava mosaic Malawi virus, East African cassava mosaic Zanzibar virus and South African cassava mosaic virus (Stanley et al., 2005; Bull et al., 2006). Although cassava begomoviruses are naturally transmitted by the whitefly Bemisia tabaci (Storey & Nichols, 1938; Dubern, 1994), CMD is widely disseminated by the distribution of stem cuttings used for vegetative propagation. Furthermore, cassava is often infected with more than one begomovirus that can increase symptom severity (synergism) and encourage virus diversification as a consequence of recombination. For example, the severe outbreak of CMD that was first reported in Northern Uganda in the 1990s and that has since spread throughout the country and to neighbouring countries in central and eastern Africa (Gibson et al., 1996; Legg et al., 2001, 2004; Neuenschwander et al., 2002; Bigirimana et al., 2004; Legg & Fauquet, 2004; Were et al., 2004; Bull et al., 2006) has been attributed to a synergistic interaction between African cassava mosaic virus (ACMV) and the East African cassava mosaic virus (EACMV) recombinant strain EACMV-UG (Deng et al., 1997; Zhou et al., 1997; Pita et al., 2001) coupled with an increase in the populations of the vector B. tabaci (Legg et al., 2002; Colvin et al., 2004).
Cultivated cassava is believed to be the principal reservoir for CMD-associated begomoviruses because of its perennial growth and scale of production (Fauquet & Fargette, 1990). However, alternative hosts have been identified (Bock et al., 1978, 1981), including Manihot glaziovii Müll. Arg., a wild relative of cassava native to Brazil (Allem, 1999) that was introduced into Africa as a potential source of resistance to pests and diseases (Allem, 2001, 2002). M. glaziovii is common in Uganda, where it is used by farmers as a source of latex and shade in crop plantations and many homesteads because of its tree-like and bushy growth. Although considered to be epidemiologically unimportant (Fauquet & Fargette, 1990) there is a high incidence of a severe mosaic disease in M. glaziovii (Fig. 1) closely resembling that associated with CMD in cassava, suggesting begomovirus aetiology. Hence, it is possible that the begomoviruses which have caused such a devastating epidemic in the highly susceptible varieties grown in Uganda can also infect M. glaziovii causing severe CMD-like disease. Despite this, there have been no reports of begomoviruses isolated from M. glaziovii or related wild species, and their possible role in the epidemiology of the disease remains unclear. Without empirical data on the begomoviruses associated with this disease and their phylogenetic and pathogenic relationships, it is impossible to assess the potential risk to cassava cultivation posed by the relatively small number of diseased M. glaziovii.
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; Zhou et al., 1997; Otim-Nape et al. 2001; Pita et al., 2001; Owor et al., 2004; Sseruwagi et al., 2004a). Recently, a comprehensive survey of cassava begomoviruses associated with CMD was undertaken in neighbouring Kenya (Bull et al., 2006, 2007). Here, we report a similar survey carried out in Uganda over the same period and have extended the investigation to include M. glaziovii as well as cassava. Cloned genomic components of representative begomovirus isolates have been constructed and used to confirm infectivity in cultivated cassava and M. glaziovii and to screen cassava cultivars for susceptibility to the disease. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and Sseruwagi et al. (2004b)] were obtained from naturally infected plants showing symptoms of CMD. Samples were taken from farmers' fields in 28 of the major growing districts in Uganda between December 2001 and December 2002. M. glaziovii plants with either white or red petioles, showing severe and mild CMD-like symptoms, respectively (Fig. 1
), were sampled from farmers' fields, homesteads and from the wild in Rakai and Wakiso districts of Uganda in July 2003. Cuttings were planted in compost and maintained at 25 °C with supplementary lighting to provide a 16 h photoperiod under containment at the John Innes Centre (UK). Virus isolates and clones were maintained and manipulated in accordance with DEFRA licence PHL 185A/4538 (7/2003).
Viral DNA extraction, cloning and analysis.
DNA was extracted using a Nucleon PhytoPure kit (Amersham) and the presence of begomovirus DNA was confirmed by PCR amplification using universal primers specific to the DNA-A component (Briddon & Markham, 1994). RFLP patterns were established for the PCR fragments using restriction enzymes DraI, EcoRV and MluI as described by Bull et al. (2006). Specific primers were subsequently designed to PCR-amplify full-length copies of the genomic components (Supplementary Table S1, available in JGV Online). PCR products were cloned into TOPO pCR2.1 according to the manufacturer's recommendations (Invitrogen). For M. glaziovii samples, extracted DNA was analysed initially by blot hybridization using full-length genomic components of EACMV-UG and ACMV as probes, labelled using a Random Primers DNA Labelling kit (Gibco-BRL) and a NucTrap Probe Purification Column (Stratagene). This was to ascertain the presence of begomovirus prior to PCR-RFLP analysis and cloning. Nucleotide sequences were established using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems) and primers designed to the vector and specific to the viral DNAs. Reaction mixtures were resolved either on an ABI 3700 automated sequencer at the John Innes Centre or commercially (Lark Technologies). Nucleotide sequences were assembled and analysed using version 7 of the Genetics Computer Group (GCG) package (Devereux et al., 1984) and aligned using CLUSTAL_X (Thompson et al., 1997). Phylogenetic analyses were performed using the neighbour-joining option of PHYLIP (version 3.57c) (Felsenstein, 1995) and phylogenetic trees were viewed and manipulated using TreeView (Page, 1996).
Infectivity of cloned viral DNA components and transmission of progeny virus.
Infectivity of cloned begomovirus components was assessed using full-length DNA-A and DNA-B excised from the cloning vector with the appropriate restriction enzyme. Combinations of DNA components were coated onto gold particles (Garzón-Tiznado et al., 1993) and introduced into Nicotiana benthamiana, cultivated cassava varieties from NACRRI, Uganda (Ebwanateraka, Nase 4, Nase 12, Njule Omumyufu, Njule Omweru and TME 14), and CIAT, Colombia (N Mex 55), and M. glaziovii by biolistic inoculation using a hand-held gun as described previously (Briddon et al., 1998). Cassava and M. glaziovii plants used for infectivity assays were produced from cuttings of healthy stock plants shown to be free of begomovirus infection by PCR analysis. Inoculated plants were maintained at 25 °C with a 16 h photoperiod.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The RFLP analysis demonstrated that the vast majority of isolates (347) corresponded to EACMV, 19 isolates corresponded to ACMV and 19 isolates were mixtures of both begomoviruses (Fig. 2b). EACMV was found in samples from all districts under investigation and the major EACMV RFLP pattern (EA1) predominated in every district. In contrast, ACMV appears to occur more sporadically and was found mainly in samples from the western half of the country. Nonetheless, both EACMV and ACMV were found in regions of the country that had experienced the CMD epidemic for different durations (Fig. 2c). Viral DNA was also extracted from M. glaziovii plants (white petioles, tree-like growth habit, severe symptoms) as well as from less severely affected plants with red petioles that had a more bushy growth habit, probably a hybrid between M. glaziovii and cassava (Fig. 1). PCR-RFLP analysis revealed two polymorphic groups corresponding to EACMV (EA1) and ACMV (A1) (data not shown), suggesting that isolates of these distinct begomovirus species are also present in the wild relative of cassava and their hybrids.
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) were selected, and full-length copies of the DNA-A and DNA-B components were cloned and characterized by sequence analysis (Table 1). Comparison of the DNA-A components from M. esculenta revealed the presence of two clusters showing >98 % (12 sequences) and 97 % identity (two sequences) between isolates within each cluster and 72–74 % identity between clusters. Comparison with published begomovirus sequences showed that the major cluster was most closely related to EACMV-UG isolates (for example, clone 24-ug177-10 shows 99.8 % identity with the Kenyan isolate EACMV-UG[K233]) and the minor cluster to ACMV isolates (for example clone 22-ug56b5 shows 97.2 % identity with the Ugandan isolate ACMV-UGMld). Hence, on the basis of accepted demarcation criteria (Stanley et al., 2005), these begomoviruses should be regarded as isolates of these two species. ACMV and EACMV-UG were also isolated from M. glaziovii plants growing in the southern districts of Rakai and Wakiso (Table 1). Phylogenetic analysis of the DNA-A components (Fig. 3a) is consistent with the proposed taxonomic status of the begomoviruses isolated from both cassava and M. glaziovii, showing that all EACMV-UG isolates are much more closely related to each other than are the ACMV isolates.
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) clustered them with the Group A sequences previously defined by Bull et al. (2006). This cluster consists of DNA-B components associated with isolates of EACMV, EACMV-UG and East African cassava mosaic Kenya virus (EACMKV) that are present in Uganda, and western and central Kenya. The latest DNA-B sequences are most closely related to EACMV-UG isolates occurring in western Kenya along the border with Uganda (for example, all four clones show >97 % identity with the Kenyan isolate EACMV-UG[K90]).
Although isolates of both species differed mainly by a series of nucleotide substitutions dispersed throughout the entire genome, the DNA-B component of EACMV-UG[Nak:1] noticeably has a 14 nt deletion located within the intergenic region upstream of the BC1 open reading frame, corresponding to nucleotides 2488–2501 in the other isolates examined. With the exception of EACMV-UG[Bus:289] DNA-A, which has a single nucleotide deletion within the coding sequence of the replication-associated protein (Rep), all DNA-A and DNA-B components have a genetic arrangement typical of Old World geminiviruses (Stanley et al., 2005).
Clones representative of all EACMV-UG RFLP patterns (EA1–EA6) were recovered in addition to a single clone (25-ug128-2) that differed from the most abundant pattern EA1 by the absence of a DraI site resulting from a single nucleotide substitution (Table 1). An ACMV clone corresponding to RFLP pattern A1 was also isolated, although clones 22-ug56b5 and wcass4-3 differed from patterns A1 and A2, respectively, by nucleotide substitutions within either MluI or DraI sites. This reinforces the contention that, although RFLP analysis can rapidly provide a general view of the population, establishment of the complete nucleotide sequence of the genomic components is essential for definitive begomovirus identification. While the majority of plants contained a single begomovirus, the nucleotide sequence data confirmed that some cassava plants supported a dual infection of EACMV-UG and ACMV. A map showing the distribution of cassava-infecting begomoviruses in Uganda in 2001–2002, based on the RFLP patterns (Fig. 2) and nucleotide sequence data (Table 1), is presented in Fig. 4.
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), although marked differences in symptom severity were associated with ACMV and EACMV-UG isolates in N. benthamiana. ACMV-[Kal:226] induced mild chlorosis and stunting symptoms in this host while all EACMV-UG isolates were more virulent, causing severe leaf curling and stunting (Fig. 5a). However, the EACMV-UG isolates were distinguishable on the basis of their slightly different latent periods (Table 2). Interestingly, N. benthamiana plants inoculated with a mixture of ACMV-[Kal:226] and EACMV-UG[Kal:233] showed more severe symptoms than either virus alone (Fig. 5b). Moreover, the presence of both viruses resulted in a higher infectivity (Table 3). Enhanced symptom severity was also observed when EACMV-UG[Kal:233] was co-inoculated with ACMV-[Kal:226] DNA-A. However, in the reciprocal combination, co-inoculation of ACMV-[Kal:226] with EACMV-UG[Kal:233] DNA-A resulted in only mild symptoms typical of ACMV infection. Co-inoculation of EACMV-UG[Kal:233] DNA-A and ACMV-[Kal:226] DNA-B or the reciprocal combination did not result in infection, consistent with the inability of the DNA-A component of one virus to trans-replicate the DNA-B component of the other as predicted from their differing Rep-binding iteron motifs, namely GGGGG (EACMV-UG) and GGAGA (ACMV).
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; Table 2). However, EACMV-UG[Kal:237] produced only a mild phenotype similar to that of ACMV-[Kal:226] and was associated with a long latent period.
Begomovirus isolates from cassava and M. glaziovii can infect both hosts
Despite the close relationship between the begomovirus isolates from cassava and M. glaziovii, it was important to confirm that viruses from one host could infect the other, an observation that would have an impact on our understanding of the epidemiology of the disease. To investigate this, plants were inoculated with the M. glaziovii isolate EACMV-UG[Wak:2] and the cassava isolate EACMV-UG[Kal:233]. The infectivity of EACMV-UG[Wak:2] was confirmed in N. benthamiana, in which it induced severe leaf curl and stunting symptoms similar to those associated with EACMV-UG[Kal:233]. EACMV-UG[Wak:2] also induced mosaic and leaf curl symptoms in its natural host M. glaziovii, similar to those observed in the field, by 30 days post-inoculation (Fig. 5c; Table 2). Furthermore, this isolate produced symptoms of severe mosaic and leaf distortion in cassava (variety N Mex 55) that were indistinguishable from those associated with EACMV-UG[Kal:233] infection in this host. In the reciprocal inoculation, EACMV-UG[Kal:233] was able to infect M. glaziovii, in which it induced symptoms similar to those associated with EACMV-UG[Wak:2]. Interestingly, EACMV-UG[Wak:2] induced severe symptoms in the highly susceptible cassava variety Ebwanateraka, although plants subsequently showed signs of recovery. However, EACMV-UG[Wak:2] was less infectious and induced less severe symptoms in cassava variety Nase 4, which contains introgressed M. glaziovii genes.
Screening cassava varieties for resistance to CMD
Cassava varieties Ebwanateraka, Njule omumyufu, Njule omweru, Nase 4, TME14 and Nase 12, known to show different responses to begomovirus infection in the field, were screened for susceptibility to CMD. Three plants of each variety were screened for disease development following biolistic inoculation of EACMV-UG[Kal:233] cloned components. Varieties Ebwanateraka and Njule omumyufu developed severe symptoms, Njule omweru and Nase 4 also developed symptoms (Fig. 5d) while TME14 and Nase 12 remained asymptomatic and viral DNA could not be detected in newly developing leaves by blot hybridization (data not shown), indicating that the experimental plants were uninfected.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Zhou et al., 1997; Otim-Nape et al., 2001; Pita et al., 2001; Sseruwagi et al., 2004a). A survey conducted during 1995 and 1996, based on PCR-amplification of DNA fragments using begomovirus-specific primers, suggested that ACMV occurred throughout the sampled areas whereas EACMV-UG occurred only in those regions that had experienced the severe epidemic (Harrison et al., 1997). A subsequent survey in 1997 was based on the use of primers to discriminate begomoviruses as well as the isolation and characterization of full-length genomic components (Pita et al., 2001). It identified ACMV and strains of EACMV, and showed that dual infections of these viruses were common, resulting in a more complex population than previously assumed as a consequence of recombination. An analysis carried out on cassava plants sampled in 2002 again identified both EACMV and ACMV, although in this instance only fragments of the viral DNA components were analysed (Sseruwagi et al., 2004a). Our present findings, based on PCR-RFLP analysis, PCR amplification of full-length genomic components and sequence analysis, are consistent with previous observations that CMD is associated with EACMV-UG and ACMV, and show that EACMV-UG is by far the predominant begomovirus throughout the entire country, consistent with the suggestion that it is displacing ACMV (Legg et al., 2006). As noted previously (Harrison et al., 1997), it is intriguing to find that EACMV-UG occurs throughout Uganda in the absence of its presumed progenitor EACMV. EACMV-UG is a recombinant virus consisting for the most part of sequences derived from EACMV, but with a small fragment of the coat protein gene derived from ACMV (Deng et al., 1997; Zhou et al., 1997) and was prevalent (in association with ACMV) during the severe epidemic of CMD that affected Uganda during the 1990s (Harrison et al., 1997). A concurrent study conducted in Kenya identified EACMV at relatively high incidence east of the Rift Valley but, for the most part, only EACMV-UG in the west of the country at the border with Uganda (Bull et al., 2006). In contrast to the findings of Were et al. (2004), who reported ACMV in western Kenya, Bull et al. (2006) found no evidence of this begomovirus in Kenya despite using protocols identical to those used in the current study to successfully detect it in Uganda. In spite of this, the overall diversity of CMD-associated begomoviruses in neighbouring Kenya (Bull et al., 2006) and Tanzania (Ndunguru et al., 2005) is significantly higher than in Uganda. This has been attributed to an ongoing repopulation of central and western Kenya by begomovirus isolates and species from coastal Kenya and Tanzania, most probably by human migration and the movement of diseased germplasm (Bull et al., 2006). Certainly, the coastal areas of Kenya have not been affected by the epidemic and neither ACMV nor EACMV-UG has been identified in that region, which otherwise harbours a genetically diverse population of CMD-associated begomoviruses. The lack of spread of the epidemic to coastal areas was likely due to geographical barriers to virus (whitefly) movement between the inland areas and the coast (Bull et al., 2006).
In this survey of 385 cassava plants, the overall frequency of co-infection with ACMV and EACMV-UG was less than 5 %. This value is significantly lower than that previously reported (>50 %) for a more limited plant sample from regions that had experienced the epidemic in 1995 (Harrison et al., 1997), consistent with EACMV-UG emerging as the dominant species in post-epidemic Uganda. Interestingly, the frequency of co-infection is also much lower than the value of 18 % reported during a comprehensive survey of post-epidemic Uganda in 2002 (Sseruwagi et al., 2004a). The reason for this is unclear, although it may simply reflect random sampling at different locations.
M. glaziovii and hybrids between M. glaziovii and cassava have been important sources of CMD resistance genes in East African cassava-breeding programmes (Bock et al., 1981; Bock & Woods, 1983). They were used to generate a number of Tropical Manihot Species (TMS) lines that were bred at IITA-Ibadan in West Africa (Legg & Fauquet, 2004; Thresh & Cooter, 2005). These improved varieties, possessing polygenic resistance to CMD derived from M. glaziovii, have been distributed widely across Africa and represent a major resource with which to combat CMD. The susceptibility of M. glaziovii to viruses associated with severe CMD raises questions about the effectiveness and long-term durability of resistance derived from this species. For example, our infectivity studies demonstrated that the cassava variety Nase 4 (also known as SS4), which derives from parental lines produced from an interspecific cross with M. glaziovii and deemed CMD resistant (Sserubombwe et al., 2001; Thresh & Cooter, 2005), is nonetheless susceptible to EACMV-UG infection. Contrary to previous assumptions, therefore, M. glaziovii might be a reservoir host for begomoviruses that could contribute to maintenance of the disease. Hence, it would be prudent to remove mosaic-affected M. glaziovii plants growing in the vicinity of cassava plants to be used as disease-free planting material.
Harrison et al. (1997) showed that cassava plants containing ACMV exhibited mild symptoms while those infected with EACMV-UG or co-infected with ACMV and EACMV-UG produced severe symptoms. They reproduced the symptom phenotype by graft inoculation of healthy cassava plants with scions from plants containing known viruses. Sseruwagi et al. (2004b) also found that ACMV occurred more frequently in mildly affected plants. In this study, we have extended these investigations using infectious cloned components and demonstrated that Ugandan isolates of ACMV and EACMV-UG generally produce mild and severe phenotypes, respectively, in both N. benthamiana and cassava. Furthermore, we have demonstrated that N. benthamiana co-infected with ACMV and EACMV-UG develops symptoms that are more severe than those induced by either virus alone. Using ACMV and East African cassava mosaic Cameroon virus (EACMCV), a distinct CMD-associated begomovirus from Cameroon, Vanitharani et al. (2004) demonstrated that synergism is due to the provision by each virus of a distinct suppressor of post-transcriptional gene silencing (PTGS), an RNA-mediated host defence response against pathogens (Voinnet et al., 1999; Voinnet, 2001). Additionally, despite ACMV and EACMV-UG being functionally incompatible due to their differing iteron sequences, we have shown that the presence of ACMV DNA-A serves to enhance EACMV-UG symptom severity whereas the reciprocal combination, EACMV-UG DNA-A in the presence of ACMV, induces only mild symptoms typical of ACMV. The presence of all viral components in systemically infected tissues in these experiments is consistent with the ability of the movement proteins encoded by DNA-B of one virus to mediate the systemic spread of the autonomously replicating DNA-A component of a second distinct virus, as demonstrated by Frischmuth et al. (1993). The symptom phenotype of bipartite begomoviruses is defined largely by the BC1 protein encoded by DNA-B (von Arnim & Stanley, 1992; Pascal et al., 1993; Ingham et al., 1995), consistent with the mild and severe phenotypes observed in these co-inoculation experiments. The enhanced symptom phenotype observed when EACMV-UG is co-inoculated with ACMV DNA-A suggests that the latter provides a PTGS suppressor, possibly AC4 as demonstrated for a Cameroon isolate of ACMV (Vanitharani et al., 2004). It is unclear why co-inoculation of ACMV with EACMV-UG DNA-A failed to enhance the symptoms of ACMV infection, particularly as AC2 (of EACMCV) has also been shown to function as a silencing suppressor (Vanitharani et al., 2004). However, this might simply reflect differences in DNA-A gene functions between isolates from different geographical locations and indicate that the ACMV DNA-B genes determine the phenotype for this particular combination.
One of the main objectives of our concurrent studies in Uganda (reported here) and Kenya (Bull et al., 2006, 2007) has been to provide clones representative of all CMD-associated begomovirus species and strains present in East Africa for use in the resistance breeding programme. Undoubtedly, the use of biolistic inoculation of cloned components has advantages over conventional field screening procedures; it allows results to be obtained more rapidly and a wider range of defined virus species and strains can be screened, since it does not rely on chance infections by locally occurring begomoviruses in the field. New cassava varieties often have to be screened at numerous locations to challenge the resistance with a representative range of species and strains, an approach that is limited by natural variation in the incidence and severity of the disease as well as its genetic makeup. Here, we have demonstrated the feasibility of a clone-based approach for screening germplasm for resistance to the disease. We have shown that several so-called resistant lines, including Nase 4 derived from TMS germplasm, are in fact susceptible to EACMV-UG, while TME14 and Nase 12 lines remained uninfected, although it should be emphasized that only a limited number of test plants were challenged on this occasion. This small-scale experiment shows that biolistic inoculation could provide a useful tool for preliminary screening for resistance, requiring relatively little material and thus overcoming time-consuming propagation required to produce sufficient material for a standard field screen. In addition, a new line can be screened against a wide range of defined species and strains to assess resistance at a single location, providing a distinct advantage over conventional screening procedures. The clones produced during this study and the concomitant study in Kenya (Bull et al., 2006, 2007) will be made available for screening purposes.
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ACKNOWLEDGEMENTS |
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Received 5 December 2007;
accepted 19 March 2008.
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