East African Cassava Mosaic Zanzibar Research Articles

East African cassava mosaic Zanzibar virus

This datasheet on East African cassava mosaic Zanzibar virus covers Identity, Distribution, Hosts/Species Affected, Vectors & Intermediate Hosts, Further Information.

First Report of East African Cassava Mosaic Virus-Uganda Infecting the Nile Tulip Tree in Western Kenya

Whitefly-transmitted cassava mosaic begomoviruses (CMBs; family Geminiviridae; genus Begomovirus) are viruses with bipartite genomes (components referred to as DNA-A and DNA-B) containing a conserved (between the two components) noncoding sequence known as the common region (CR) required for DNA replication and transcription (Fondong 2013). CMBs cause cassava mosaic disease (CMD), a major limitation to cassava production in sub-Saharan Africa. In Kenya, CMD is associated with the begomoviruses East African cassava mosaic virus (EACMV), African cassava mosaic virus (ACMV), East African cassava mosaic Kenya virus, and East African cassava mosaic Zanzibar virus, as well as several recombinant viruses, such as the virulent EACMV-Uganda (EACMV-UG) that so far occurs only in western Kenya (Nyongesa Were et al. 2016). Within the CMB-cassava pathosystem, various noncassava plant species may play an epidemiological role as virus reservoirs for the whitefly vector Bemisia tabaci (Alabi et al. 2008). In May 2017, leaf samples were collected from three plants exhibiting virus-like symptoms including mosaic, mottling, twisted leaves, and reduction in leaf surface area as well as from two nonsymptomatic plants belonging to the family Bignoniaceae growing in three cassava fields in western Kenya. Total genomic DNA was extracted from leaf samples using the ZR Plant/Seed DNA MiniPrep kit as per the manufacturer’s instructions (Zymo Research Corp., U.S.A.). The identity of the plant species was confirmed as Markhamia lutea by phenotypic characteristics and by polymerase chain reaction (PCR) amplification, Sanger sequencing, and BLASTn analysis of the chloroplast rubisco large-chain gene using the primer pair RbcLa-F (5′-ATGTCACCACAAACAGAGACTAAAGC-3′)/RbcLa-R (5′-GTAAAATCAAGTCCACCRCG-3′). Samples were screened by PCR with the diagnostic primer pairs JSP001/JSP002 for ACMV, JSP001/JSP003 for EACMV (Fondong et al. 2000), and UV AL1-F1/ACMV CP-R3 for EACMV-UG (Zhou et al. 1997). Samples from nonsymptomatic plants did not yield amplification products, confirming absence of viral infection. A 1,650-bp fragment of the expected size was amplified from one symptomatic sample with the primer pair UV AL1-F1/ACMV CP-R3, suggesting the presence of EACMV-UG. To confirm the positive PCR result, the genome of the virus was enriched by rolling-circle amplification (RCA) using a TempliPhi 100 RCA Kit (GE Healthcare, U.S.A.). The library was prepared using the NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, U.S.A.) according to the manufacturer’s instructions and sequenced using an Illumina MiSeq platform with the MiSeq Reagent Kit version 3 (600 cycles) (Illumina, U.S.A.). Following de novo sequence assembly, 344 and 89 paired-end reads aligned to generate two contigs identified by BLASTn search as the DNA-A and DNA-B of a bipartite virus. The nucleotide sequences were deposited in GenBank as accession numbers MF974568 and MH379641. The 2,798-nucleotide (nt) DNA-A and 2,774-nt DNA-B had an approximately 160-nt EACMV-like CR with 53%-nt sequence identity. DNA-A shared 99% nucleotide sequence identity with the DNA-A of an EACMV-UG isolate (AJ717522) from western Kenya, whereas the DNA-B was closely related to another EACMV-UG isolate (AJ704962) from western Kenya at 97% nucleotide sequence identity. To the best of our knowledge, this is the first report of occurrence of EACMV-UG in a species of the family Bignoniaceae. Further investigations are required to determine the epidemiological significance of M. lutea in the CMB-cassava pathosystem.

Status of cassava mosaic begomoviruses in farmers’ fields in Ghana

A survey for cassava mosaic disease (CMD) was carried out in Ghana from 2007−2008 to determine the status of cassava mosaic begomoviruses in farmers’ fields. The survey covered cassava growing areas in five major cassava producing regions of Ghana. Out of 136 fields visited, the plants in 5% were not affected by CMD, 18% contained plants with mild symptoms, whereas 77% had cassava with moderately severe or severe symptoms. A total of 412 cassava leaf samples and a symptomatic Manihot glaziovii sample were analyzed using polymerase chain reaction. African cassava mosaic virus (ACMV) alone was detected in 42.0% of symptomatic cassava leaves with the remaining 58% being mixed infected by ACMV and East African cassava mosaic virus (EACMV). Mixed ACMV and EACMV infections were detected in symptomatic M. glaziovii, two non-symptomatic cassava samples and in individual whitefly vectors. EACMV was not detected alone in any cassava or whitefly sample. South African cassava mosaic virus (SACMV), Indian cassava mosaic virus (ICMV), East African cassava mosaic Zanzibar virus (EACMZV) and the Uganda strain of EACMV were not detected in any cassava or whitefly sample. The occurrence of high proportion of mixed infections of cassava by cassava mosaic begomoviruses (CMBs), which could lead to emergence of new species or variants in the country, require concerted effort to mitigate the CMD problem.

A single-tube duplex and multiplex PCR for simultaneous detection of four cassava mosaic begomovirus species in cassava plants

A single-tube duplex and multiplex PCR was developed for the simultaneous detection of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Malawi virus (EACMMV) and East African cassava mosaic Zanzibar virus (EACMZV), four cassava mosaic begomoviruses (CMBs) affecting cassava in sub-Saharan Africa. Co-occurrence of the CMBs in cassava synergistically enhances disease symptoms and complicates their detection and diagnostics. Four primer pairs were designed to target DNA-A component sequences of cassava begomoviruses in a single tube PCR amplification using DNA extracted from dry-stored cassava leaves. Duplex and multiplex PCR enabled the simultaneous detection and differentiation of the four CMBs, namely ACMV (940bp), EACMCV (435bp), EACMMV (504bp) and EACMZV (260bp) in single and mixed infections, and sequencing results confirmed virus identities according to the respective published sequences of begomovirus species. In addition, we report here a modified Dellapotra et al. (1983) protocol, which was used to extract DNA from dry and fresh cassava leaves with comparable results. Using the duplex and multiplex techniques, time was saved and amount of reagents used were reduced, which translated into reduced cost of the diagnostics. This tool can be used by cassava breeders screening for disease resistance; scientists doing virus diagnostic studies; phytosanitary officers checking movement of diseased planting materials, and seed certification and multipliers for virus indexing.

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.

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.

First report of the occurrence of East African cassava mosaic virus‐Uganda (EACMV‐UG) in Angola

Cassava mosaic disease (CMD) is the major production constraint on cassava ( Manihot esculenta ) in Angola. Previous reports derived from single samples recorded the presence of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) in Angola. Regional CMD monitoring initiatives have predicted the spread into northern Angola of EACMV-Uganda (-UG), the unusually virulent (aggressive) recombinant strain associated with the African CMD pandemic (Legg et al ., 2006). In view of this threat, an assessment survey was conducted in north and central Angola in April 2008. Eighteen cassava fields were sampled in Bengo, Cuanza Norte, Malanje and Uige Provinces. Moderate to severe CMD symptoms were observed in most fields. Sixty-three of 65 samples obtained from these fields tested positive in TAS-ELISA using monoclonal antibody SCR 20. PCR was performed using primer pairs previously described for the specific identification of ACMV, EACMV, EACMV-UG, East African cassava mosaic Cameroon virus (EACMCV) and Indian cassava mosaic virus (ICMV) (Ogbe et al ., 2003), East African cassava mosaic Malawi virus (EACMMV) (Zhou et al ., 1998), East African cassava mosaic Zanzibar virus (EACMZV) (Were et al ., 2004) and South African cassava mosaic virus (SACMV) in mixed infections. This revealed the occurrence of ACMV, EACMV and EACMV-UG, but not EACMZV, EACMMV, SACMV or ICMV. Single infections of ACMV and EACMV were detected in 32·3 and 9% of samples, respectively. Mixed infections of ACMV with EACMV, EACMV with EACMV-UG and all three viruses were detected in 35·3, 1·5 and 16·9% of samples, respectively. ACMV, detected in 84·6% of samples, was the predominant species, followed by EACMV (63%) and EACMV-UG (18·5%). Significantly, EACMV-UG occurred most frequently in the northernmost part of the surveyed area (Uige Province) immediately to the south of the Bas Congo region of the Democratic Republic of Congo, already known to be affected by the pandemic. These findings significantly broaden the known geographical extent of the CMD pandemic and draw attention to the urgent need for the large-scale deployment of resistant cassava varieties, which have been used to reduce losses in pandemic-affected regions of East Africa.

Application of a Simplified Molecular Protocol to Reveal Mixed Infections with Begomoviruses in Cassava

AbstractSamples of cassava leaves exhibiting severe symptoms of cassava mosaic disease (CMD) were collected with the PhytoPASS kit in fields surrounding the city of Bujumbura (Burundi). These materials were then sent to Belgium for polymerase chain reaction determination of the CMD begomoviruses inducing the observed symptoms. Different pairs of specific primers were used to amplify DNA sequences specific to 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), the Uganda variant of East African cassava mosaic virus (EACMV‐UG) and South African cassava mosaic virus (SACMV). It was revealed that mixed infections were prevailing in the analyzed materials. Most of the samples submitted to this analysis were found to be co‐infected by three different begomoviruses (ACMV + EACMV + EACMV‐UG). The so revealed mixed infections could explain the high severity of CMD symptoms noticed on cassava in the region of Bujumbura while the diversity within the CMD causal agents illustrates the importance to take this parameter into consideration for a successful use of plant genetic resistance to control the disease.

Diversity of begomoviruses associated with mosaic disease of cultivated cassava (Manihot esculenta Crantz) and its wild relative (Manihot glaziovii Müll. Arg.) in Uganda

Cassava (Manihot esculenta) growing in Uganda during 2001-2002 has been screened for the presence of begomoviruses using PCR-RFLP, cloning full-length genomic components and nucleotide sequence analysis. In contrast with a recent survey in neighbouring Kenya, which identified three distinct strains of East African cassava mosaic virus (EACMV, EACMV-UG and EACMV-KE2) as well as East African cassava mosaic Zanzibar virus and the new species East African cassava mosaic Kenya virus, only EACMV-UG and, to a lesser extent, African cassava mosaic virus (ACMV) were found associated with cassava in Uganda. The integrity of the cloned genomic components of representative virus isolates was confirmed by demonstrating their infectivity in Nicotiana benthamiana and cassava using biolistic inoculation, providing a convenient means to screen cassava varieties for disease resistance. Both EACMV-UG and ACMV were also associated with Manihot glaziovii. Infectivity studies using cloned components confirmed that viruses from one host could infect the other, suggesting that this wild relative of cassava might be a reservoir host for the disease. The relatively low level of diversity of begomoviruses associated with cassava mosaic disease in Uganda is consistent with reports that EACMV-UG has displaced other begomovirus species and strains during the recent epidemic that swept through the country.

Infectivity, pseudorecombination and mutagenesis of Kenyan cassava mosaic begomoviruses

Cloned DNA-A and DNA-B components of Kenyan isolates of East African cassava mosaic virus (EACMV, EACMV-UG and EACMV-KE2), East African cassava mosaic Kenya virus (EACMKV) and East African cassava mosaic Zanzibar virus (EACMZV) are shown to be infectious in cassava. EACMV and EACMKV genomic components have the same iteron sequence (GGGGG) and can form viable pseudorecombinants, while EACMZV components have a different sequence (GGAGA) and are incompatible with EACMV and EACMKV. Mutagenesis of EACMZV has demonstrated that open reading frames (ORFs) AV1 (encoding the coat protein), AV2 and AC4 are not essential for a symptomatic infection of cassava, although mutants of both ORF AV1 and AV2 produce attenuated symptoms in this host. Furthermore, ORF AV1 and AV2 mutants were compromised for coat protein production, suggesting a close structural and/or functional relationship between these coding regions or their protein products.

Genetic diversity and phylogeography of cassava mosaic viruses in Kenya

Cassava is a major factor in food security across sub-Saharan Africa. However, the crop is susceptible to losses due to biotic stresses, in particular to viruses of the genus Begomovirus (family Geminiviridae) that cause cassava mosaic disease (CMD). During the 1990s, an epidemic of CMD severely hindered cassava production across eastern and central Africa. A significant influence on the appearance of virus epidemics is virus diversity. Here, a survey of the genetic diversity of CMD-associated begomoviruses across the major cassava-growing areas of Kenya is described. Because an initial PCR-restriction fragment-length polymorphism analysis identified a much greater diversity of viruses than assumed previously, representative members of the population were characterized by sequence analysis. The full-length sequences of 109 components (68 DNA-A and 41 DNA-B) were determined, representing isolates of East African cassava mosaic virus and East African cassava mosaic Zanzibar virus, as well as a novel begomovirus species for which the name East African cassava mosaic Kenya virus is proposed. The DNA-B components were much less diverse than their corresponding DNA-A components, but nonetheless segregated into western and eastern (coastal) groups. All virus species and strains encountered showed distinct geographical distributions, highlighting the importance of preventing both the movement of viruses between these regions and the importation of the disease from adjacent countries and islands in the Indian Ocean that would undoubtedly encourage further diversification.

Occurrence and distribution of cassava begomoviruses in Kenya

SummaryA survey for cassava mosaic disease (CMD) was conducted in Kenya, to investigate the factors contributing to the generally increased incidence and severity of CMD in the cassava growing regions and to study the distribution of the disease's causal begomoviruses, African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) and their strains. Special emphasis was given to the occurrence of the destructive recombinant Uganda variant strain of EACMV (EACMV‐UG2). Samples from 91 farmers' fields in the main cassava‐growing areas of coastal and western Kenya were collected and subjected to ELISA and PCR for detection and typing of the begomoviruses present. CMD incidence was highest in western Kenya (80–100%) and lowest in the Coast province (25–50%). In Western and Nyanza provinces, 52% of the samples tested contained EACMV‐UG2, 22% ACMV and 17% contained both ACMV and EACMV‐UG2. EACMV was found in four cases at different sites. In cassava samples from the coast province, only EACMV with DNA‐A sequences similar to EACMV strains present in Kenya and Tanzania was found. East African cassava mosaic Zanzibar virus (EACMZV) was present in several farms in the Kilifi district. In 15% of all cassava samples with CMD symptoms, flexuous, filamentous virus‐like particles were also found, providing evidence for a more complex virus situation in cassava grown at the Kenyan coast. In western Kenya, where intense cassava cultivation takes place, CMD is rampant and EACMV‐UG2 was found in mixed virus infections with ACMV driving the epidemics. In coastal areas, where farms are scattered and in isolation, EACMV is endemic, however, with a lower disease incidence and with a limited impact to cassava production.

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

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).

Variations and taxonomic status of begomoviruses causing severe epidemics of cassava mosaic disease in Kenya, Uganda, and Democratic Republic of the Congo

The molecular variability in the DNA-A of cassava-begomoviruses, East African cassava mosaic virus (EACMV), East African cassava mosaic virus – Uganda variant (EACMV-UG), African cassava mosaic virus (ACMV), and East African cassava mosaic Zanzibar virus (EACMZV) in Kenya, Uganda, and Democratic Republic of the Congo (DRC) was investigated. Most samples from western Kenya, Uganda, and eastern DRC contained EACMV-UG. Fewer than half of the samples had a mixed infection of ACMV and EACMV-UG, and a small percentage had only ACMV infections. EACMV and EACMZV were the only begomoviruses detected in samples from the Kenyan coast. The coat protein gene nucleotide (nt) and the deduced amino acid sequence analyses revealed a high degree of sequence identity within each virus type and that EACMV-UG was most related to ACMV. However, analysis of the retrieved complete DNA-A (2781–2801 nt) sequences of selected virus types revealed that EACMV-UG DNA-A share more than 90% identity with EACMV and less than 80% with ACMV, confirming that the virus is a strain of EACMV.

Occurrence of East African cassava mosaic Zanzibar virus (EACMZV) in coastal Kenya

a Department of Disease and Stress Biology, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK; b Kenya Agricultural Research Institute, PO Box 57811, Nairobi, Kenya; c Plant, Animal & Human Health Group, Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, UK; and d DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmBH, Plant Virus Division, Biologisches Bundesanstalt, Messeweg 11/12, D-38104 Braunschweig, Germany