A novel cassava-infecting begomovirus from Madagascar: cassava mosaic Madagascar virus

Cassava mosaic geminiviruses (CMGs) are implicated in cassava mosaic disease (CMD), the main constraint to cassava production in Africa. Here, we report the complete nucleotide sequences of the DNA-A and DNA-B of a newly characterized CMG found infecting cassava in Madagascar, for which we propose the tentative name cassava mosaic Madagascar virus. With the exception of two recombinant regions that resembled a CMG, we determined that the non-recombinant part of the DNA-A component is distantly related to the other CMGs. Whereas the DNA-B component possesses one recombinant region originating from an unidentified virus, the rest of the genome was seen to be closely related to members of the species East African cassava mosaic Zanzibar virus (EACMZV). Phylogenetic analysis based on complete genome sequences demonstrated that DNA-A and DNA-B components are outliers related to the clade of EACMV-like viruses and that DNA-A is related to the monopartite tomato leaf curl begomoviruses described in islands in the south-west Indian Ocean.     

Molecular evidence for diverse populations of cassava-infecting begomoviruses in southern Africa

Summary.  In this paper we describe results obtained from screening field populations of begomoviruses from infected cassava from six countries in southern Africa. It was found that African cassava mosaic virus occurs in five of the six countries (except Angola), East African cassava mosaic virus is present in five countries (except Zambia) and South African cassava mosaic virus was detected only in South Africa and Swaziland. In addition, we report for the first time in southern Africa, the appearance of the Ugandan variant virus (UgV) which was found frequently in mixed infections with other cassava-infecting begomoviruses. Accepted May 24, 2001 Received March 5, 2001     

Biodiversity and recombination of cassava-infecting begomoviruses from southern India

Summary. Cassava mosaic disease (CMD) is caused by various begomoviruses of the family Geminiviridae leading to considerable crop losses in Africa and Asia. Recombination between their genomic components has generated new pathotypes with enhanced virulence in Africa. Here, we report about a survey on the biodiversity of begomoviruses in cassava from southern India (Tamil Nadu and Kerala states) performed in 2001 and 2002. Viral DNA A components from stem cuttings were analysed using polymerase chain reaction and restriction fragment length polymorphism. Eight representative examples were completely sequenced. The majority of DNA sequences (7 of 8) obtained were more closely related to that of Sri Lankan cassava mosaic virus (SLCMV) than of Indian cassava mosaic virus (ICMV). Only one sequence collected in Kerala was related to ICMV. The diversity of the SLCMV-like sequences was rather low compared to the variability of African viruses associated with cassava mosaic disease. Based on DNA A sequence data, all of these isolates should be classified as variants of SLCMV or ICMV. Phylogenetic analysis revealed mosaic structures within the DNA sequences which may indicate footprints of recombination events between ancestors of SLCMV and ICMV.     

Dual infection by cassava begomoviruses in two leguminous species (Fabaceae) in Yangambi, Northeastern Democratic Republic of Congo

A study on cassava mosaic begomoviruses was conducted around Yangambi (DR Congo) by sampling 10 different leguminous species with or without symptoms similar to cassava mosaic disease. DNA was isolated to amplify CMBs using primers targeting AC2 and AC4 genes for virus detection by PCR. The results showed a dual infection by ACMV and EACMV in two weed species, Centrosema pubescens and Pueraria javanica, associated with mosaic symptoms. The DNA-A genome component of ACMV and EACMV from the infested weeds was sequenced. Seven ACMV and four EACMV isolates are reported. The major ACMV strains were closely related to ACMV-NGogo, ACMV-IC and ACMV-UGMld, whereas all EACMV strains were closely related to a Uganda variant, the most prevalent virus. This study shows that whiteflies may transmit CMBs to non-cassava plants under high epidemic pressure.     

Alternate hosts of African cassava mosaic virus and East African cassava mosaic Cameroon virus in Nigeria

Cassava mosaic disease (CMD) caused by African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) is the major constraint to cassava production in Nigeria. Sequences of the DNA-A component of ACMV and EACMCV isolates from leguminous plant species (Senna occidentalis, Leucana leucocephala and Glycine max), castor oil plant (Ricinus communis), a weed host (Combretum confertum) and a wild species of cassava (Manihot glaziovii) were determined. All ACMV isolates from these hosts showed 96-98% nucleotide sequence identity with cassava isolates from West Africa. EACMCV was found only in four hosts (S. occidentalis, L. leucocephala, C. confertum, M. glaziovii), and sequences of these isolates showed 96-99% identity with cassava isolates from West Africa. These results provide definitive evidence for the natural occurrence of ACMV and EACMCV in plant species besides cassava.     

Genetic variability of East African cassava mosaic Cameroon virus under field and controlled environment conditions

Cassava geminiviruses occur in all cassava growing areas of Africa and are considered to be the most damaging vector-borne plant pathogens. At least seven species of these viruses have been identified. We investigated genetic variation in East African cassava mosaic cassava Cameroon virus (EACMCV) from naturally infected cassava and from experimentally infected Nicotiana benthamiana. Results showed that the populations of EACMCV in cassava and in N. benthamiana were genetically heterogeneous. Mutation frequencies in the order of 10⁻⁴, comparable to that reported for plant RNA viruses, were observed in both hosts. We also produced an EACMCV mutant that induces reversion and second site mutations, thus suggesting that a high mutation frequency facilitates the maintenance of genome structure and function. This is direct experimental evidence showing that cassava geminiviruses exhibit a high mutation frequency and that a single clone quickly transforms into a collection of mutant sequences upon introduction into the host.     

East African cassava mosaic Zanzibar virus – a recombinant begomovirus species with a mild phenotype

Summary. Cassava plants exhibiting mild symptoms of cassava mosaic disease (CMD) were collected from Unguja island, Zanzibar. Cuttings grown from these plants in the glasshouse produced similar symptoms, which were milder than those caused by other known cassava mosaic geminiviruses (CMGs). The whitefly vector, Bemisia tabaci (Gennadius), transmitted the putative virus to 27.7% (n=18) of target plants. Total DNA extracted from diseased leaves did not yield diagnostic PCR-bands using virus-specific primers to known CMGs. Degenerate primers, however, produced a diagnostic band indicating the presence of a begomovirus. Full-length DNA-A (2785 nucleotides) and DNA-B (2763 nucleotides) components were subsequently PCR-amplified, cloned and sequenced. Phylogenetic analyses of DNA-A and -B sequences showed that they were most similar to strains of East African cassava mosaic virus from Tanzania and Uganda at 83% and 86% nucleotide identities, respectively. The number and arrangement of open reading frames were similar to those of bipartite begomoviruses from the Old World. DNA-A was predicted to have recombined in the intergenic region (IR), AC1 and AC4 genes, and DNA-B in the IR. A maximum nucleotide identity of 83% in the DNA-A component with other sequenced begomoviruses, together with different biological properties allows this virus to be recognised as belonging to a new species named East African cassava mosaic Zanzibar virus (EACMZV).     

Co-adaptation between cassava mosaic geminiviruses and their local vector populations

Four cassava mosaic geminivirus (CMG) isolates; African cassava mosaic virus from Namulonge, Uganda (ACMV-[Nam]), East African cassava mosaic virus from Mtwara, Tanzania (EACMV-[Mtw]), EACMV-Uganda from Namulonge (EACMV-UG[Nam]) and Indian cassava mosaic virus from Trivandrum, India (ICMV-[Tri]) were compared for their ability to be transmitted by four colonies of cassava whitefly, Bemisia tabaci (Gennadius), collected from Namulonge (NAM), Mtwara (MTW), Kumasi (KUM) and Trivandrum (TRI). With 20 adult whiteflies per test plant, the CMGs from Africa were transmitted by all three of the African B. tabaci populations to 60-79% of the target plants. Indian cassava B. tabaci transmitted ICMV-[Tri] to 89% of the target plants. In contrast, Indian cassava B. tabaci transmitted EACMV-[Mtw] and Tanzanian cassava B. tabaci transmitted ICMV-[Tri] less than a tenth as often, even when using 50 adults per plant and with increased acquisition and inoculation access periods. The complete coat protein genes of the CMGs had sequences typical of their source viruses, the major differences occurring between those originating from India and Africa. Symptom severity of the CMGs was quantified precisely by both visual assessment and image analysis techniques. EACMV-[Mtw] and ACMV-[Nam] were the most and least damaging isolates with 15.4 and 10.0% of the leaf area diseased, respectively. In these tests the transmission frequency was not linked to symptom severity in the source plants. These data support the hypothesis that virus-vector co-adaptation exists in the cassava mosaic disease (CMD) pathosystem and the results are discussed in relation to CMD epidemiology.     

Infectivity of African cassava mosaic virus clones to cassava by biolistic inoculation

Summary.  Clones of an African cassava mosaic virus isolate originating from Nigeria (ACMV-NOg) were shown to be infectious to cassava by biolistic inoculation. The production of pseudorecombinants between ACMV-NOg and clones of an ACMV isolate originating from Kenya (ACMV-K) indicated that the lack of infectivity of ACMV-K to cassava was due to defect(s) in the DNA B genomic component; this component encodes two proteins involved in cell-to-cell movement. This is the first demonstration of infectivity of a cloned geminivirus to cassava and conclusively proves that ACMV is the causative agent of cassava mosaic disease. The potential uses of infectious ACMV clones and the means by which to introduce them into cassava are discussed. Received January 18, 1998 Accepted May 27, 1998     

Introduction of East African cassava mosaic Zanzibar virus to Oman harks back to “Zanzibar, the capital of Oman”

Cassava mosaic disease (CMD) is the most devastating disease of the subsistence crop cassava (Manihot esculenta) across Africa and the Indian subcontinent. The disease is caused by viruses of the genus Begomovirus (family Geminiviridae)—seven species have been identified so far. The Sultanate of Oman is unusual among countries in Arabia in growing cassava on a small scale for local consumption. During a recent survey in A’Seeb wilayat of Muscat governorate, Oman, cassava plants were identified with symptoms typical of CMD. A begomovirus, East African cassava mosaic Zanzibar virus (EACMZV), was isolated from symptomatic plants. This virus was previously only known to occur in Zanzibar and Kenya. During the 19th Century, Zanzibar was governed by Oman and was so important that the Sultan of Oman moved his capital there from Muscat. After a period of colonial rule, the governing Arab elite was overthrown, following independence in the 1960s, and many expatriate Omanis returned to their homeland. Having gained a liking for the local Zanzibar cuisine, it appears that returning Omanis did not wish to do without dishes made from one particular favorite, cassava. Consequently, they carried planting material back to Oman for cultivation in their kitchen gardens. The evidence suggests that this material harbored EACMZV. Recently, Oman has been shown to be a nexus for geminiviruses and their associated satellites from diverse geographic origins. With their propensity to recombine, a major mechanism for evolution of geminiviruses, and the fact that Oman (and several other Arabian countries) is a major hub for trade and travel by air and sea, the possibility of onward spread is worrying.     

Role of a geminivirus AV2 protein putative protein kinase C motif on subcellular localization and pathogenicity

Virus-derived genes or genome fragments are increasingly being used to generate transgenic plants with resistance to plant viruses. There is need to rapidly investigate these genes in plants using transient expression prior to using them as transgenes since they may be pathogenic to plants. In this study, we investigated the AV2 protein encoded by East African cassava mosaic Cameroon virus, a virus associated with a cassava disease epidemic in western Africa. For subcellular localization, AV2 was fused to the yellow fluorescent protein (YFP) and expressed in Nicotiana benthamiana. Confocal analyses showed that AV2-YFP localizes mainly in the cytoplasm. Because it overlaps with the coat protein gene and therefore could be used to generate transgenic plants for resistance to geminiviruses, we investigated its pathogenesis in N. benthamiana by using the Potato virus X (PVX) vector. The chimeric virus PVX-AV2 induced a mild mottling in infected plants and was shown to suppress virus-induced gene silencing (VIGS). Using point mutations, we show here that AV2 pathogenicity is dependent on a conserved putative protein kinase C (PKC) phosphorylation motif. Because of its pathogenicity and ability to suppress RNA silencing, AV2 transgenic plants will less likely provide a control to geminiviruses, indeed it may weaken the resistance of the plant. We therefore suggest the use of the AV2 putative PKC mutants to generate transgenic plants.     

Both Indian cassava mosaic virus and Sri Lankan cassava mosaic virus are found in India and exhibit high variability as assessed by PCR-RFLP

Summary. The biodiversity of geminiviruses associated with the Cassava Mosaic Disease (CMD) in India was investigated using PCR to specifically amplify the DNA of Indian cassava mosaic virus (ICMV) or Sri Lankan cassava mosaic virus (SLCMV) and also by using PCR to amplify specific viral genes, followed by digestion with different restriction endonucleases to obtain polymorphic patterns (PCR-RFLP). Results showed that both ICMV and SLCMV were present in mosaic-affected cassava; ICMV was geographically restricted to certain regions, whereas SLCMV was widespread. PCR-RFLP analysis showed that, in addition to ICMV-type and SLCMV-type patterns, a high proportion (40%) of the samples displayed novel patterns, some of which were localized in certain areas, whereas others were widely distributed.     

Characterization of tobacco geminiviruses in the Old and New World

Summary.  Biological differences and molecular variability between six phenotypically distinct tobacco-infecting geminivirus isolates from southern Africa (Zimbabwe) and Mexico were investigated. Host range studies conducted with tobacco virus isolates ZIM H from Zimbabwe and MEX 15 and MEX 32 from Mexico indicated all had narrow host ranges restricted to the Solanaceae. Alignment of coat protein gene (CP) and common region (CR) sequences obtained by PCR, and phylogenetic analysis of the CP sequences indicated Zimbabwean isolates were distantly related to those from Mexico and that geographically proximal isolates shared their closest affinities with Old and New World geminiviruses, respectively. Zimbabwean isolates formed a distinct cluster of closely related variants (>98% sequence identity) of the same species, while MEX 15 segregated independently from MEX 32, the former constituting a distinct species among New World geminiviruses, and the latter being a variant, Texas pepper virus-Chiapas isolate (TPV-CPS) with 95% sequence identity to TPV-TAM. Results collectively indicated a geographic basis for phylogenetic relationships rather than a specific affiliation with tobacco as a natural host. MEX 15 is provisionally described as a new begomovirus, tobacco apical stunt virus, TbASV, whose closest CP relative is cabbage leaf curl virus, and ZIM isolates are provisionally designated as tobacco leaf curl virus, TbLCV-ZIM, a new Eastern Hemisphere begomovirus, which has as its closest relative, chayote mosaic virus from Nigeria. Received August 6 1998 Accepted October 22, 1998     

The use of biolistic inoculation of cassava mosaic begomoviruses in screening cassava for resistance to cassava mosaic disease

Inoculation of cassava with infectious clones of cassava mosaic geminiviruses (Geminiviridae: Begomovirus) and total DNA extracts from plants infected with well-characterised viruses was evaluated using the Bio-Rad Helios Gene Gun System. Total DNA extracts from infected plants and cloned viruses were produced for coating gold particles and bombardment onto new cassava genotypes, 96/1089A, 96/1039, 96/0160, 96/0304 and three local landraces TME 117, TME 3 and TME 4. Cloned DNA of a Kenyan isolate of the recombinant variant of East African cassava mosaic virus (EACMV-UG2-[Ka]), was only infectious to TME 117 (7/10 plants), 3 weeks post-inoculation with mild infection symptoms in the newly developing leaves. Biolistic inoculation with a chimeric pseudorecombinant virus between DNA A and B components from EACMV-[Ke-Kilifi] and EACMV-UG2-[Ka], respectively, was infectious to TME 117, 96/1039 and 96/0304 and developed very severe and persistent symptoms. TME 3 and TME 4 also developed symptoms, 12 days post-inoculation (d.p.i.). Total DNA extracts of ACMV and EACMV-[Ke-Kilifi] resulted in serious infections with symptoms already evident, 10d.p.i. In general, biolistic inoculation trials with total DNA extracts resulted in a higher number of infected plants expressing symptoms at a much earlier stage (10-12d.p.i.) compared with trials inoculated with virus clones.     

Molecular characterization of a new alphasatellite associated with a cassava mosaic geminivirus in Madagascar

Two complete nucleotide sequences of an alphasatellite isolated from a cassava plant with mosaic disease symptoms in Madagascar are described and analyzed. While the helper begomovirus was identified as an isolate of East African cassava mosaic Kenya virus (EACMKV), its associated alphasatellite was most closely related (80 % nucleotide sequence identity) to cotton leaf curl Gezira alphasatellite. These satellite molecules have typical features of alphasatellites, with a single gene in the virion sense, an A-rich region and a stem-loop structure. According to the proposed species demarcation threshold of alphasatellites (83 % nucleotide identity), they are isolates of a new species for which we propose the name “Cassava mosaic alphasatellite”.     

Cassava mosaic virus disease in East Africa: a dynamic disease in a changing environment

Cassava mosaic disease (CMD), now known to be caused by cassava mosaic geminiviruses (Family Geminiviridae; Genus Begomovirus), was first reported in East Africa in 1894. Epidemics occurred in Madagascar and Uganda in the 1930s and 1940s, and more localised rapid spread of CMD was observed in parts of coastal Tanzania in the 1930s and coastal Kenya in the 1970s. During the 1990s, a major regional pandemic of an unusually severe form of CMD has expanded to affect parts of at least five countries, causing massive economic losses and destabilising food security. Mechanisms responsible for the development and progress of the pandemic have been described, and comparisons of epidemiological data for varieties grown throughout the period under review suggest that the recent pandemic has been characterised by rapid rates of CMD spread hitherto unknown in East Africa. A key factor in the genesis and spread of the pandemic has been the recombination between two distinct cassava mosaic geminiviruses to produce a novel and more virulent hybrid. Although such events may be common, the known history of CMD in East Africa suggests that the frequency with which they become epidemiologically significant is low. A corollary of this is that resistance, developed originally in Tanzania between 1934 and 1960, and utilized and supplemented at the International Institute of Tropical Agriculture, Nigeria, since 1971, is providing effective CMD control in current pandemic-affected areas of East Africa. Consequently, it is concluded that prospects for managing CMD in the 21st century are good, and that the approach adopted should build on the model of collaborative research and implementation that has been established in tackling the current CMD pandemic.     

Biolistic infection of cassava using cloned components of Indian cassava mosaic virus

Summary. Cassava mosaic disease (CMD) is a major constraint to cassava production in Africa and Asia. Of the two begomoviruses associated with CMD on the Indian subcontinent, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus, only the latter has been successfully reintroduced into cassava to resolve the aetiology of the disease. Here, we report the complete nucleotide sequence of an ICMV isolate from Maharashtra (ICMV-[Mah2]), central India. Biolistic inoculation of the cloned components produced a systemic infection and typical mosaic symptoms in cassava, thereby fulfilling Koch’s postulates. The availability of infectious clones will provide a valuable tool to screen new cassava cultivars for disease resistance under defined conditions.     

Short deletions in nuclear targeting sequences of African cassava mosaic virus coat protein prevent geminivirus twinned particle formation

Coat proteins (CPs) of geminiviruses are multifunctional proteins. Using transient expression experiments, we have recently identified putative sequence motifs of African cassava mosaic virus (ACMV) CP involved in nuclear import (NLS) and export (NES) (Virology 286 (2001) 373). Here, we report on the effect of corresponding deletion mutants in the context of infecting viruses. Since NLS and NES may overlap with DNA binding and multimerisation domains, we have investigated their effect on viral infection, particularly, on particle formation. All deletion mutants were infectious in Nicotiana benthamiana when co-inoculated with DNA B, but poorly sap-transmissible. Some of the mutants showed reduced levels of viral single-stranded DNA (ssDNA), whereas the amount of double-stranded DNA (dsDNA) was not greatly affected. None of these CP mutants was able to produce stable virus particles. In contrast, viruses with CP fused to Flag epitopes at the N- or C-terminus (CP:Flag or Flag:CP) were readily sap-transmissible and formed amorphous nucleoprotein particles but only few geminate structures. The relevance of the identified sequences in replicating viruses with reference to nuclear import and export as well as to particle stability and DNA binding is discussed.