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1 Department of Disease and Stress Biology, John Innes Centre (JIC), Colney, Norwich NR4 7UH, UK
2 Kenya Agricultural Research Institute, Katumani Applied Biotechnology Laboratory, PO Box 340, Machakos, Kenya
Correspondence
John Stanley
john.stanley{at}bbsrc.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ704934–AJ704974 and AJ717516–AJ717583.
Present address: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK. 
Present address: Plant Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Jhang Road, Faisalabad, Pakistan.
Present address: Department of Plant Science and Crop Protection, University of Nairobi, PO Box 30197 Nairobi, Kenya.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and Asia (Hong et al., 1993; Saunders et al., 2002; Dutt et al., 2005).
Cassava mosaic viruses, in common with all geminiviruses, have single-stranded DNA genomes that are encapsidated in characteristic twinned (so-called geminate) particles. The genomes of the majority of begomoviruses consist of two components, termed DNA-A and DNA-B (Stanley et al., 2005). The DNA-A component encodes proteins required for viral DNA replication [the replication-associated protein (Rep) that initiates replication and the replication-enhancer protein (REn)], control of gene expression [the transcriptional activator protein (TrAP), required for initiating transcription of the virion-sense genes and also involved in suppression of post-transcriptional gene silencing-mediated host defences; Voinnet et al., 1999; Vanitharani et al., 2005] and insect transmission [the coat protein (CP) that is vital for plant-to-plant transmission by the whitefly Bemisia tabaci]. The functions of two other DNA-A-encoded proteins (AV2 and AC4) remain unclear, although possible roles in movement and pathogenicity/suppression of post-transcriptional gene silencing, respectively, have been demonstrated. The DNA-B component encodes the nuclear shuttle protein (NSP) and movement protein (MP) that act co-operatively to move the virus both within and between cells in host plants (reviewed by Hanley-Bowdoin et al., 2004).
Members of six species of begomovirus have been identified in association with CMD in Africa: African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV), East African cassava mosaic Zanzibar virus (EACMZV) and South African cassava mosaic virus (SACMV) (reviewed by Legg & Fauquet, 2004). These viruses are believed to have evolved from indigenous African viruses that adapted to cassava upon its introduction during the 18th century. Initially they existed in distinct geographical regions (Hong & Harrison, 1995), but more recently their distribution has become more complex as a result of two factors. Firstly, cassava is propagated vegetatively, a process that not only perpetuates the virus, but also leads to dissemination of the virus in infected planting material (Gibson & Otim-Nape, 1997; Chellappan et al., 2004). Both trade and human migration due to drought and conflict can result in the spread of infected planting material over great distances. Secondly, the occurrence of CMD epidemics, the most recent of which initiated in or around northern Uganda during the 1990s, swept though Uganda and surrounding countries and continues to affect cassava throughout central Africa (Legg & Fauquet, 2004). The severe CMD phenotype that is associated with this epidemic is caused by the synergistic interaction between ACMV and a distinct recombinant strain of EACMV [EACMV-Uganda (EACMV-UG)], commonly known as the ‘Uganda variant’ (Zhou et al., 1997; Pita et al., 2001).
Representatives of only three begomovirus species have previously been reported from Kenya, namely ACMV, EACMV and EACMZV (Stanley & Gay, 1983; Bull et al., 2003; Were et al., 2004a, b). EACMV and the recombinant strain EACMV-UG have been identified in the east and west of the country, respectively. EACMZV is a recently identified begomovirus species that is believed to have originated on the island of Zanzibar, but is now also found in coastal areas of Kenya (Bull et al., 2003; Maruthi et al., 2004). We have surveyed all major cassava-growing areas of Kenya to assess the current diversity and geographical distribution of begomoviruses associated with CMD. This study has identified a novel begomovirus species and a new recombinant strain of EACMV, and illustrates the increasing diversity and geographical distribution of these pathogens in this region. Such information will be useful in devising measures aimed at reducing the impact of CMD and ensuring that breeding programmes produce resistant cassava varieties that target relevant virus species and strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The plants were grown in peat-based compost in insect-proof glasshouses maintained at 25 °C with supplementary lighting to provide a 16 h photoperiod.
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, which was designed to amplify all but 17 nt of the DNA-A component of begomoviruses, was used to amplify viral DNA for RFLP analysis. The products obtained were digested with restriction enzymes DraI, EcoRV and MluI (Invitrogen) and visualized on ethidium bromide-stained agarose gels by using a UV transilluminator. For amplification of full-length components, specific, partially overlapping primers were designed to aligned sequences (data not shown) of cassava begomoviruses available in GenBank. For DNA-A, virion-sense primer EAFL01 (5'-GTCGACGTCATCAATGACGTTGTACCAGGCG-3') and complementary-sense primer EAFL02 (5'-GTCGACCCCCACTACCTCAAACACTTCAAAG-3') included unique SalI sites (underlined). For DNA-B, two pairs of specific primers were designed, both of which included a unique SstI site (underlined): virion-sense primer FLCB01 (5'-GAGCTCGGAAATTGGAGATTGATTGGTGC-3') with complementary-sense primer FLCB02 (5'-GAGCTCATTCGAGCAGGCGACCATGCG-3'), and virion-sense primer FLCB03 (5'-GCTTAGAGCTCGGAAATTGGAGATTGATTGGTGC-3') with complementary-sense primer FLCB04 (5'-AAAGGGAGCTCATTCGAGCAGGCGACCATGCG-3'). PCR amplification was conducted as described previously (Briddon & Markham, 1994) with annealing temperatures ranging from 45 to 50 °C.
Cloning, sequencing and sequence analysis.
Potentially full-length PCR products were cloned into the vector pCR2.1-TOPO and transformed into One Shot TOP10 competent Escherichia coli cells as recommended by the manufacturer (Invitrogen). DNA sequencing was conducted by using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer). Reaction products were resolved commercially (Lark Technologies, UK). Sequence information was stored, assembled and analysed by using the Genetics Computer Group program suite (version 7) (Devereux et al., 1984) and sequence alignments were produced by using CLUSTAL_X (Thompson et al., 1997). Phylogenetic analyses were conducted on matrices of aligned sequences by using the neighbour-joining and bootstrap options of PHYLIP (version 3.5c) (J. Felsenstein, Department of Genetics, University of Seattle, WA, USA). Dendrograms were viewed, manipulated and printed by using TreeView (Page, 1996). The aligned sequences were analysed for possible recombination events by using RDP (version 2) (Martin & Rybicki, 2000).
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RESULTS |
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), a number of which did not correspond to the predicted RFLP patterns of previously identified virus sequences available in GenBank. This level of diversity was considerably higher than that found in a similar study undertaken at the same time in Uganda (unpublished results). The DNA-A and DNA-B components of a number of representative viruses were cloned and sequenced in their entirety in an attempt to resolve the restriction patterns and assign them to known or previously unidentified cassava mosaic virus species/strains.
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; Fig. 2). These sequences are available in GenBank under the accession numbers given in Table 1. The components were all of typical begomovirus size, ranging from 2784 nt (isolates K3, K5 and K270) to 2802 nt (isolate K36). The majority of the clones had predicted genes typical of Old World begomoviruses in terms of size and position. However, for a number of clones, some of the predicted genes contained a premature stop codon, frame-shift mutation or upstream in-frame initiation codon, as indicated in Table 1.
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). They all had lengths typical of begomovirus DNA-B components, ranging from 2743 nt (isolate K18) to 2776 nt (isolates K90 and K298). The majority of the sequences encode the two oppositely orientated genes typical of DNA-B components. However, clones obtained from isolates K72 and K197 both contained a frame-shift mutation in the complementary-sense gene, resulting in a truncated MP coding sequence.
Phylogenetic analysis of DNA-A components
A phylogenetic dendrogram of the complete DNA-A sequences of the new begomovirus isolates, as well as other CMD-associated begomoviruses available in GenBank, is shown in Fig. 3. Many of the viruses characterized here group with previously identified species/strains of cassava begomoviruses from Kenya, namely EACMV-UG, EACMV and EACMZV, but not with EACMCV, EACMMV and SACMV that are found elsewhere in Africa. However, the dendrogram highlights two groups of previously unidentified viruses. The first group comprises a novel strain of EACMV that we shall refer to as EACMV-KE2. The DNA-A components of the nine EACMV-KE2 isolates are related closely to, but distinct from, previously identified EACMV isolates. The EACMV-KE2 sequences show approximately 98 % nucleotide sequence identity to EACMV, but only 88 % identity to EACMZV, indicating that they should be considered as strains of EACMV. However, alignment of the sequences of EACMV-KE2[K48] with those of EACMV-[K24] and EACMZV-[K18], which are representative of these clusters, show a small region of the Rep gene (highlighted in the PLOTSIMILARITY analysis of Fig. 4a) that is related more closely to the analogous region of EACMZV. This region, between nt 1887 and 2085, has 97 % nucleotide sequence identity to EACMZV and only 88 % identity to EACMV. Thus, EACMV-KE2 DNA-A is a recombinant consisting for the most part of sequences derived from EACMV, but with a small fragment of the Rep coding sequence probably originating from EACMZV. Interestingly, EACMZV is also a recombinant consisting of sequences derived from EACMV, but with 5'-terminal Rep coding sequences and part of the intergenic region derived from an as-yet-unidentified begomovirus (Maruthi et al., 2004). The region of sequence exchanged within EACMV-KE2 encodes approximately 65 aa of Rep and spans the Walker A motif (GXXXXGKT) involved in ATP binding (Walker et al., 1982). It does not affect the downstream sequences encoding the B and C motifs (Gorbalenya et al., 1990) or the upstream sequences encoding amino acids involved in additional Rep functions (reviewed by Hanley-Bowdoin et al., 1999).
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). Overall, the EACMKV DNA-A sequences have approximately 84 % nucleotide sequence identity to EACMV-UG, 87 % to EACMV, 88 % to SACMV and 89 % to EACMZV. PLOTSIMILARITY comparisons of the nucleotide sequence of EACMKV-[K261] with those of EACMV-[K24] and SACMV are presented in Fig. 4(b). Between nt 2143 and 2797, EACMKV-[K261] shows 92 % identity to the analogous region of SACMV, but only 69 % identity to that of EACMV-[K24] (and EACMZV-[K18]). In contrast, nt 1–986 of EACMKV-[K261] show 97 % identity to EACMV-[K24] (and EACMZV-[K18]), but only 78 % to SACMV. A further breakdown was done to try to determine whether this section of sequence is derived from EACMV or EACMZV. However, nt 1–193 show 96 % identity to both EACMZV-[K18] and EACMV-[K24] (and only 90 % to SACMV), and nt 194–986 show 97 % identity to both EACMZV-[K18] and EACMV-[K24] (and only 77 % identity to SACMV). Although this sequence analysis is inconclusive, we believe that the recombinant is more likely to be derived from EACMV, given its geographical origin (as discussed later). Thus the DNA-A component of EACMKV is a recombinant consisting of sequences probably originating from EACMV [nt 1–986; region A in Fig. 4(b)] and SACMV [nt 2143–2797; region C in Fig. 4(b)]. The sequences spanning the remaining region [nt 987–2142; region B in Fig. 4(b)] are largely indistinguishable between strains of EACMV and SACMV (95 % identity to EACMZV-[K18], 95 % to SACMV and 91 % to EACMV-[K24]). The likely origins of the sequences constituting EACMKV are further highlighted in the phylogenetic dendrogram derived from the alignment of AC4 amino acid sequences (Fig. 5). This analysis shows the sequence of EACMKV to be more similar to that of SACMV than to those of EACMV, EACMZV or ACMV. As the recombinant fragment of EACMKV involves the 5' terminus of the Rep gene, it is not surprising that the dendrogram based on Rep amino acid sequences also shows grouping of the EACMKV isolates with SACMV. For the remaining genes, EACMKV sequences are indistinguishable from those of EACMV, EACMZV and EACMV-KE2 (for example, dendrograms based on alignments of the amino acid sequences of the CP and AV2 are shown in Fig. 5).
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and will not be elaborated on here.
Phylogenetic analysis of full-length DNA-B components
A phylogenetic dendrogram comprising the complete DNA-B sequences of the new begomovirus isolates, as well as other CMD-associated begomoviruses, is shown in Fig. 3. As with the analysis of DNA-A components, no DNA-B component clones corresponding to either ACMV or EACMCV were identified in this study. No sequences of the DNA-B components of EACMMV are available in GenBank for comparison at this time. Overall, the sequences of the DNA-B components of EACMV, EACMZV and SACMV, which form a tight cluster, are much less variable than those of their DNA-A counterparts. The DNA-B components of ACMV and EACMCV are distinct from, and basal to, this cluster.
Two major groups of EACMV-like DNA-B sequences are evident. The first (indicated as group A in Fig. 3) consists of the DNA-B components of isolates originating from central and western locations, comprising most of the EACMV-UG isolates, two EACMV isolates (K29 and K268) and all of the EACMKV isolates. Interestingly, the DNA-B component of SACMV clusters with, and is basal to, this group. SACMV has recently been shown to be present not only in South Africa, but also in Madagascar and Zimbabwe (Ranomenjanahary et al., 2002; Briddon et al., 2004), but apparently not in Tanzania (Ndunguru et al., 2005) or Kenya (Were et al., 2004a, b; this study). The group A DNA-B sequences additionally segregate into two clusters indicated as subgroups A1 and A2 (Fig. 3). Subgroup A1 consists of those components isolated from samples collected in districts bordering Uganda and central Kenya, whilst subgroup A2 consists of components originating from isolates collected in districts bordering Tanzania and central Kenya (subgroup A2). The second major group (indicated as group B in Fig. 3) consists of virus isolates originating mainly from central and eastern (coastal) areas of Kenya, comprising the majority of the EACMV isolates, all of the EACMV-KE2 isolates, two EACMV-UG isolates (K66 and K72, both of which originate from close to the border with Uganda) and all of the EACMZV isolates (including that originating from Zanzibar). The components associated with EACMV-UG[K66] and [K72] that do not fit with the east–west phylogenetic grouping may be derived from an east Kenyan non-EACMV-UG isolate that pseudorecombined with EACMV-UG following its recent introduction into the region bordering Uganda. Two subgroups of group B isolates (indicated as subgroups B1 and B2 in Fig. 3) are evident. Subgroup B1 comprises all of the EACMV-KE2 isolates, the majority of the EACMV isolates and two EACMV-UG isolates. Subgroup B2 consists entirely of EACMZV isolates. Although the differences between group A and group B DNA-B components are dispersed throughout the entire sequence, the majority of changes are situated in the common (non-coding) region immediately upstream of the conserved geminivirus stem–loop structure (results not shown). The subgroup B1 components maintain the iteron (Rep-binding motif) of the group A components (GGGGG). However, the remaining non-coding sequence is more typical of subgroup B2. By comparison, the predicted iteron motif of EACMZV (subgroup B2) is GGAGA.
Phylogeographical analysis
The viruses identified during this project have distinct, but frequently overlapping, geographical distributions (Fig. 2). The preponderance of EACMV-UG isolates originate in districts neighbouring Uganda and, to a lesser extent, central Kenya; there is no evidence for EACMV-UG in coastal districts. Previous studies have similarly been unable to identify EACMV-UG in coastal regions of either Kenya or adjacent Tanzania (Were et al., 2004a; Ndunguru et al., 2005). EACMV is prevalent in coastal and central districts of the country, but apparently only at low incidence in western areas. It is possible that EACMV in western Kenya was largely displaced by EACMV-UG, believed to be a more virulent pathogen, with only pockets of EACMV remaining. The newly identified recombinant EACMV-KE2 appears to be restricted to the coastal districts of Kwale and Malindi, although it was not identified in the intervening district of Kilifi. EACMZV was identified throughout the coastal district as well as in the central districts of Machakos and Makueni, although there are currently no reports of its spread southwards into Tanzania. EACMKV was identified in two areas, in the central district of Machakos and along the Tanzania/Kenya border close to Lake Victoria (Kuria, Migori and Suba districts). The isolates from these areas form two related clusters in the phylogenetic tree, indicating that they have recently diverged and are probably evolving independently.
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DISCUSSION |
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; Sseruwagi et al., 2004), including Uganda (unpublished results), were hindered by the unexpected levels of diversity encountered in the begomovirus population associated with CMD in Kenya. This finding led us to characterize the viruses by sequence analysis of full-length clones, which not only provided diversity data, but also allowed us to assign the PCR-RFLP patterns to species/strains. However, the RFLP map generated from the full-length DNA-A component sequence did not always reflect the population of virus as determined by PCR-RFLP analysis. For example, EACMV-UG isolates K137 and K139 are represented by RFLP class 6 (Fig. 1), but their DNA-A sequences indicate DraI profiles corresponding to class 4. This RFLP class also contains EACMV-UG[K90], although its sequence would predict the establishment of a distinct class. This implies a certain degree of variation within each isolate population that is not necessarily represented by the cloned full-length DNA-A components. In the majority of cases, the diagnostic restriction sites were gained or lost by a single nucleotide change. For example, RFLP profiles of EACMZV isolates K18 (class 2) and K19 (class 3) are distinguishable by a single nucleotide change within an EcoRV site (Fig. 1b). However, this behaviour did not change the species to which the isolate was assigned and confirms that there is some sequence variation for a single virus isolate within a plant. While this highlights a major drawback of the approach, our PCR-RFLP analysis does indicate that co-infections between species are considerably lower than have been reported for other studies. The reason for this is unclear, but may suggest that a barrier exists (possible cross-protection) between some CMD species. Most cases of co-infection in cassava involve EACMV-UG and ACMV, this association being responsible for the CMD epidemic. The synergism between these two virus species is believed to be due to a selective advantage conferred by each partner providing an essential factor, which leads to a severe phenotype. Specifically, the two viruses have been shown to provide differentially acting suppressors of post-transcriptional gene silencing that are required to overcome host defences (Vanitharani et al., 2004). This suggests that, in the absence of a synergistic interaction, only one virus in a co-infected plant will become predominant and be maintained. Our results indicate that, although technically less demanding, cheaper and quicker than sequencing, PCR-RFLP may have only limited use for future virus characterization in Kenya because of the complexity of the begomovirus population. In addition, our analysis demonstrates that sequence determination of the CP (and AV2) coding region, which is frequently used to characterize begomoviruses (Brown et al., 2001) and has proved useful for identifying the majority of CMD-associated species, is inadequate for identifying EACMV strains (with the exception of EACMV-UG, due to its recombinant CP gene) or for distinguishing between EACMV, EACMKV and EACMZV.
Previous studies have shown ACMV, EACMV, EACMV-UG and EACMZV to be present in Kenya. With the exception of ACMV, our investigation confirms these earlier reports and shows that EACMV, EACMV-UG and EACMZV have distinct geographical distributions. Surprisingly, ACMV was not identified in Kenya during the course of our study. This conflicts with an early report by Stanley & Gay (1983) and another, more recent study by Were et al. (2004b), who showed the presence of ACMV in western Kenya during 1999 at relatively high incidence (39 %) in CMD-affected plants, either as single infections or co-infections with EACMV-UG2. It is unlikely that the PCR primers used in our study were unable to detect ACMV, as its components have been amplified successfully from samples originating in Uganda (unpublished data), Nigeria (Briddon & Markham, 1994) and archival ACMV-infected cassava samples originating from Kenya (unpublished data). It is possible that our sampling procedure was biased against ACMV-infected plants, which may have a mild-symptom phenotype, by selecting only severely affected plants in each location. However, our sampling procedure was not significantly different from other studies that identified ACMV in Kenya. Therefore, it is far more likely that the lack of identification of ACMV is due to a very low incidence of this species in the country at the time of sampling, possibly due to displacement by EACMV-UG associated with the CMD epidemic.
Each of the virus species and strains identified shows a distinct geographical distribution within Kenya. EACMZV is believed to have originated on the island of Zanzibar, which lies off the Tanzania coast, and introduced to coastal areas of Kenya via infected planting material (Bull et al., 2003; Maruthi et al., 2004). The virus is now prevalent in coastal districts of Kenya and also occurs in pockets inland. The spread inland is more likely to be due to the transport of planting material along the main Mombassa to Nairobi road rather than by insect transmission. However, in view of its apparently rather limited interaction with other begomoviruses, it is likely that EACMZV represents a relatively recent introduction. EACMV, considered to be representative of a parental non-recombinant species associated with CMD, was identified predominantly in coastal and central areas, and apparently is absent from districts along the Uganda border where EACMV-UG is ubiquitous. EACMV-UG, together with ACMV, is associated with the severe CMD epidemic that swept through Uganda in the 1990s and continues to affect surrounding countries (Legg & Fauquet, 2004). The prevalence of EACMV-UG in western districts would seem to suggest that it has displaced the other CMD-associated begomoviruses and strains. However, EACMV-UG is not present in coastal districts. As CMD is disseminated through cuttings, this suggests that there has been little or no movement of infected planting material eastwards. In addition, the geographical barrier imposed by a non-cultivated, arid lowland area between the coastal strip and the central highlands may prevent the movement of viruliferous insects between the two areas. This barrier may also have prevented the spread of the newly identified EACMV-KE2 further inland. The fact that its proposed parental viruses EACMV and EACMZV are found on both sides of this arid area suggests that the recombination event to produce EACMV-KE2 has occurred only recently. Whatever the reason for the restricted movement of these viruses, it has for the moment prevented the spread of EACMV-UG, associated with the extremely destructive CMD epidemic that has swept across sub-Saharan Africa, from reaching coastal regions of Kenya.
The distribution of EACMKV at two distinct geographical regions is intriguing and suggests that there has been movement of virus between these two areas. However, this is unlikely to have occurred recently, as the virus populations are genetically distinguishable. Whilst whitefly transmission of EACMKV cannot be ruled out as the mechanism of spread, it is far more likely to be due to the transportation of infected cassava cuttings. In this case, farmers may have moved from the districts near Lake Victoria to the more affluent areas around Nairobi. It is also possible that the movement of EACMKV, as well as EACMZV, occurred immediately after the CMD epidemic. EACMZV has been reported to have a mild-symptom phenotype (although we have also found it associated with severe infections) and this species may have become established after importation of cassava by farmers replacing their varieties ravaged by the epidemic. Surprisingly, EACMKV was not identified in previous studies (Were et al., 2004a; Ndunguru et al., 2005), both of which examined the same geographical areas of Kenya or adjacent areas of Tanzania. In both cases, however, only limited numbers of clones were sequenced and the studies relied extensively on ELISA and PCR diagnostics, which would not have distinguished EACMKV from EACMV.
The results of our sequence analyses show that the DNA-B components can provide important data on the evolution and spread of these CMD-associated viruses. Previous analyses have concentrated for the most part on DNA-A sequences, possibly due to the present standard of defining begomovirus species by their complete DNA-A sequences (Fauquet et al., 2003). The DNA-B components of EACMV, EACMKV, EACMZV and SACMV are related very closely, forming an ‘east African’ cluster distinct from ACMV and EACMCV. The results show good evidence of recent intraspecific component exchange (so-called pseudorecombination) between strains of EACMV. For example, the DNA-B components of EACMV-UG isolates K66 and K72 and EACMV-KE2 (particularly EACMV-KE2[K201]) are related very closely, as are those of EACMV-UG[K137] and EACMV-[K29]. The clustering of EACMV-UG[K66] and [K72] DNA-B components, originating from the Ugandan border, with those of EACMV and EACMZV isolates from central and coastal districts is again consistent with a recent pseudorecombination event following the movement of infected planting material into post-epidemic regions in the west of the country. There is also evidence for interspecific component exchange between EACMV and EACMZV (subgroups B1 and B2 in Fig. 3). The DNA-B component of begomoviruses depends upon DNA-A-encoded Rep for trans-replication and yet the Rep-recognition sequences (iterons) of EACMV and EACMZV differ. Component exchange on this occasion has therefore required the replacement of the EACMZV DNA-B origin of replication (or at least iteron-containing sequences) with that of EACMV.
The presence of two EACMZV-like DNA-B types in Kenya, those associated with EACMZV (subgroup B2) and those associated with EACMV (subgroup B1), can be explained by two possible scenarios. Either they are the result of a single introduction of EACMZV that underwent pseudorecombination with EACMV or there were two introductions of EACMZV, the first of which led to the pseudorecombinant DNA-B associated with EACMV (but apparently not the establishment of EACMZV) and the second led to the establishment, at least in a confined area, of EACMZV. The close clustering (lack of diversity) of EACMZV DNA-B components serves to reinforce the idea that the introduction of EACMZV to Kenya has occurred relatively recently, their sequences remaining very similar to that of the isolate originating from Zanzibar (EACMZV-[TZ : Ugu]; Fig. 3). Additionally, phylogenetic trees based upon DNA-B sequences, but ignoring the common region, are largely identical to that presented in Fig. 3 (results not shown), with the subgroup B1 DNA-B components being distinct from those of subgroup B2. This indicates that the sequences have diverged and that the clustering is not due to the proposed exchange of the origin of replication. Taken together, these data suggest that there have been two separate introductions of EACMZV (or at least its DNA-B component) to Kenya, rather than a single introduction.
Our study has shown that the genetic diversity of begomoviruses associated with CMD is considerably greater than reported previously and highlights the importance of recombination in the evolution of these viruses. Identification of the range of species and strains of viruses present in a particular area provides important information for strategies to control CMD, allowing new varieties to be tested for resistance/tolerance specifically to the range of viruses identified in the planting region. We are currently investigating the infectivity of cognate DNA-A and DNA-B clones produced during the course of this work in order to assess their pathogenicity and potential use in CMD resistance-breeding programmes. Our findings also provide a platform for monitoring novel virus species and strains that evolve or are introduced. Future efforts should address the diversity of viruses in neighbouring countries, as our study, as well as other reports, indicates that novel viruses such as EACMZV and possibly EACMKV can originate elsewhere. The importance of the islands in the Indian Ocean and countries to the south contributing to the diversity of CMD-associated begomoviruses in Kenya has been highlighted here. This supports the suggestion of Ndunguru et al. (2005) that eastern Africa serves as the ‘melting pot’ for CMD begomovirus diversity and evolution from which the viruses spread throughout the cassava-growing areas of the continent.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 7 March 2006;
accepted 9 June 2006.
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