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.     

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.     

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).     

Begomoviruses from mungbeans in Pakistan: epitope profiles,DNA A sequences and phylogenetic relationships

Summary. Monoclonal antibodies raised against particles of African Cassava mosaic virus, Indian Cassava mosaic virus or Okra leaf curl virus were used to test samples collected in Pakistan from begomovirus-infected plants. Epitope profiles obtained from cucurbits resembled those previously reported for Pakistani begomoviruses. In contrast, epitope profiles obtained from legumes showed little diversity and were quite distinct from these. DNA with nucleotide sequences typical of begomovirus DNA A components was amplified from selected mungbean samples. Comparisons of the sequences of the amplified DNA with other begomovirus DNA A sequences and phylogenetic analysis revealed that the Pakistani mungbean viruses were isolates of the species Mungbean yellow mosaic India virus, which together with Mungbean yellow mosaic virus represents a distinct lineage of Old World begomoviruses.Present address: Plant Virology Laboratory, National Agricultural Research Centre, Islamabad 45500, Pakistan.     

Molecular characterisation of a distinct South African cassava infecting geminivirus

Summary.  African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) are whitefly-transmitted geminiviruses (WTGs) which are widespread in cassava in Africa and cause serious yield losses. Recently, a new geminivirus affecting cassava in South Africa (SACMV) has been reported. In this work SACMV was found to have DNA-A and DNA-B components. Comparisons of amino acid sequences of the putative coat protein, and nucleotide sequences of the common region and a 687-bp DNA B fragment of SACMV with other WTGs, showed that SACMV clustered with the Old World subgroup of the Begomovirus genus of geminiviruses. Despite its bipartite nature, SACMV was most closely related to monopartite TYLCVs, but was sufficiently different to justify designating it as a distinct virus. In serological studies, SACMV grouped biologically with EACMV isolates. Received January 22, 1998 Accepted May 21, 1998     

Identification of a second begomovirus,Sri Lankan cassava mosaic virus,causing cassava mosaic disease in India

Summary. The DNA A and DNA B components of a begomovirus associated with cassava mosaic disease (CMD) originating from Kerala, India, were cloned. Biolistically inoculated clones induced symptoms typical of CMD in cassava. Sequence comparisons showed the virus to be an isolate of Sri Lankan cassava mosaic virus (SLCMV). This is the first time this begomovirus species has been identified in India and only the second species shown to cause CMD in the country. The implication of these findings on our understanding of the diversity and geographic distribution of CMD-associated begomoviruses in the region and on efforts to obtain resistance to CMD are discussed.     

Two new ‘legumoviruses’ (genus Begomovirus) naturally infecting soybean in Nigeria

Two new ‘legumoviruses’ (genus Begomovirus; family Geminiviridae) naturally infecting soybean (Glycine max L. Merr.) in Nigeria were molecularly characterized. Based on characteristic symptoms in soybean, the two viruses are provisionally designated as Soybean mild mottle virus (SbMMV) and Soybean chlorotic blotch virus (SbCBV). SbCBV has a bipartite genome, whereas SbMMV has only a DNA A component. The DNA A component of SbMMV is 2,768 nucleotides (nt) long and the DNA A and DNA B components of SbCBV are 2,708 and 2,647 nt long, respectively. In pairwise comparisons, the DNA A component of SbMMV and SbCBV showed 62% nt sequence identity, indicating that these two viruses are distinct. Whereas the DNA A of SbMMV contains two virion- and four complementary-sense open reading frames, that of SbCBV lacks the virus-sense AV2, a signature gene present in ‘Old World’ begomoviruses. A pairwise comparison with the corresponding nucleotide sequence of other begomoviruses in the databases indicated that SbCBV had a maximum of 74% identity with cowpea golden mosaic virus and SbMMV had a maximum of 65% identity with mungbean yellow mosaic India virus and kudzu mosaic virus. Phylogenetic analysis of the DNA A component of SbCBV and SbMMV together with those of other begomoviruses available in the databases showed clustering of the two viruses within the ‘legumovirus’ clade of the begomovirus phylogenetic tree. In addition, the DNA A and B components of SbCBV from Centrosema pubescens Benth were found to be identical to those from soybean, indicating that leguminous wild species are a potential alternative host for the virus. Since soybean is an introduced crop, the identification of two distinct begomoviruses naturally infecting soybean in Nigeria suggests the occurrence of ‘legumoviruses’ in plant species indigenous to Africa and underscores their potential threat to sustainable cultivation of soybean on the African continent.     

<Emphasis Type="Italic">Sida micrantha</Emphasis> mosaic is associated with a complex infection of begomoviruses different from <Emphasis Type="Italic">Abutilon mosaic virus</Emphasis>

Summary. We report on the nucleotide sequences of geminiviruses of the genus Bemogovirus infecting Sida micrantha Schr., a common weed in Brazil. For decades, the mosaic frequently associated with Sida plants was considered to be caused by a Brazilian strain of Abutilon mosaic virus (AbMV). By infection studies and sequence comparisons, we demonstrate that it is associated with a complex of at least two begomoviruses as different from AbMV as most South American geminiviruses. Two molecules of DNA A (A1, A2) and three of DNA B (B1, B2, B3) were cloned and sequenced. According to the high homology in their common regions, DNA A1 and DNA B3, as well as DNA A2 and DNA B2, are cognate components of two begomoviruses, which were infectious in Nicotiana benthamiana plants. No trans-replication was found for any other A/B combination. The intergenic region of DNA B2 appears to be the product of the recombination between DNA B1 and DNA A2. These results show that a coinfection of begomoviruses can persist over decades, producing a reservoir of partially recombined but distinct geminiviruses.     

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.     

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.     

RNA viruses and their silencing suppressors boost Abutilon mosaic virus, but not the Old World Tomato yellow leaf curl Sardinia virus

Mixed viral infections can induce different changes in symptom development, genome accumulation and tissue tropism. These issues were investigated for two phloem-limited begomoviruses, Abutilon mosaic virus (AbMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) in Nicotiana benthamiana plants doubly infected by either the potyvirus Cowpea aphid-borne mosaic virus (CABMV) or the tombusvirus Artichoke mottled crinkle virus (AMCV). Both RNA viruses induced an increase of the amount of AbMV, led to its occasional egress from the phloem and induced symptom aggravation, while the amount and tissue tropism of TYLCSV were almost unaffected. In transgenic plants expressing the silencing suppressors of CABMV (HC-Pro) or AMCV (P19), AbMV was supported to a much lesser extent than in the mixed infections, with the effect of CABMV HC-Pro being superior to that of AMCV P19. Neither of the silencing suppressors influenced TYLCSV accumulation. These results demonstrate that begomoviruses differentially respond to the invasion of other viruses and to silencing suppression.     

Tomato mottle Taino virus pseudorecombines with PYMV but not with ToMoV: Implications for the delimitation of <Emphasis Type="Italic">cis</Emphasis>- and <Emphasis Type="Italic">trans</Emphasis>-acting replication specificity determinants

Summary. Over the last decade, the tomato production in Cuba has been affected by new whitefly-associated diseases. In addition to the well-documented presence of Tomato yellow leaf curl virus (TYLCV) along the island, the occurrence of bipartite begomoviruses has also been reported. One of them, tentatively named Tomato mottle Taino virus (ToMoTV), has now been cloned and characterized at the molecular level. Its genomic organization is similar to other bipartite geminiviruses. Phylogenetic analyses placed ToMoTV in a subcluster with other geminiviruses isolated in the Caribbean Basin: Tomato mottle virus (ToMoV), Bean dwarf mosaic virus, Abutilon mosaic virus, Sida golden mosaic virus and Potato yellow mosaic virus (PYMV). Biolistic inoculation of tobacco and tomato plants with cloned viral DNA showed that ToMoTV pseudorecombines with PYMV-GP as predicted by the identity of their iterative elements, whereas it does not show the same ability with ToMoV, even when their replication-associated proteins (Rep and REn) show the highest percentage of similarity. A comparative analysis of Rep proteins from begomoviruses that are able to produce viable reassortants suggests that some key elements for virus replication specificity are located in the first ten amino acids of this protein.Received October 18, 2002; accepted April 22, 2003 Published online July 2, 2003     

Molecular characterization and experimental host range of an isolate of <Emphasis Type="Italic">Wissadula golden mosaic St. Thomas virus</Emphasis>

Partial genome segments of a begomovirus were previously amplified from Wissadula amplissima exhibiting yellow-mosaic and leaf-curl symptoms in the parish of St. Thomas, Jamaica and this isolate assigned to a tentative begomovirus species, Wissadula golden mosaic St. Thomas virus. To clone the complete genome of this isolate of Wissadula golden mosaic St. Thomas virus, abutting primers were designed to PCR amplify its full-length DNA-A and DNA-B components. Sequence analysis of the complete begomovirus genome obtained, confirmed that it belongs to a distinct begomovirus species and this isolate was named Wissadula golden mosaic St. Thomas virus-[Jamaica:Albion:2005] (WGMSTV-[JM:Alb:05]). The genome of WGMSTV-[JM:Alb:05] is organized similar to that of other bipartite Western Hemisphere begomoviruses. Phylogenetic analyses placed the genome components of WGMSTV-[JM:Alb:05] in the Abutilon mosaic virus clade and showed that the DNA-A component is most closely related to four begomovirus species from Cuba, Tobacco leaf curl Cuba virus, Tobacco leaf rugose virus, Tobacco mottle leaf curl virus, and Tomato yellow distortion leaf virus. The putative Rep-binding-site motif in the common region of WGMSTV-[JM:Alb:05] was observed to be identical to that of Chino del tomate virus-Tomato [Mexico:Sinaloa:1983], Sida yellow mosaic Yucatan virus-[Mexico:Yucatan:2005], and Tomato leaf curl Sinaloa virus-[Nicaragua:Santa Lucia], suggesting that WGMSTV-[JM:Alb:05] is capable of forming viable pseudo-recombinants with these begomoviruses, but not with other members of the Abutilon mosaic virus clade. Biolistic inoculation of test plant species with partial dimers of the WGMSTV-[JM:Alb:05] DNA-A and DNA-B components showed that the virus was infectious to Nicotiana benthamiana and W. amplissima and the cultivated species Phaseolus vulgaris (kidney bean) and Lycopersicon esculentum (tomato). Infected W. amplissima plants developed symptoms similar to symptoms observed under field conditions, confirming that this virus is a causal agent of Wissadula yellow mosaic disease in W. amplissima.     

Molecular characterization of a distinct bipartite begomovirus species infecting tomato in India

A distinct bipartite begomovirus was found associated with tomato plants showing yellowing, curling, and crumpling of the leaves, in a sub-temperate region in India. The complete DNA-A and DNA-B components were amplified through rolling circle amplification (RCA) using Φ-29 DNA polymerase and characterized. The DNA-A of the isolate was comprised of 2,756 nucleotides, encoding six open reading frames (ORFs) and DNA-B that of 2,725 nucleotides, encoding two ORFs. Genome organization of the isolate was typical of an old world bipartite begomovirus. Comparisons showed that DNA-A and its intergenic region (IR) have the highest sequence identity (86% and 84%, respectively) with the Tomato leaf curl New Delhi virus (ToLCNDV; DQ116885) and some other begomoviruses (>84%) reported from cucurbits and tomato. This data suggested that the isolate is a distinct begomovirus species for which a name Tomato leaf curl Palampur virus (ToLCPMV) is proposed. DNA-B showed the maximum sequence identity (73%) with Tomato leaf curl New Delhi virus-India-[Pakistan:Dargai:T5/6:2001] (AY150305). The common region (CR) of DNA-A and DNA-B showed 94% sequence similarity with each other. In the present study, phylogenetic relationship of this new species was also established with different begomoviruses reported from tomato and other begomoviruses showing highest homologies with complete DNA-A and DNA-B sequences. ToLCPMV is being reported from a sub-temperate region in India which was previously unaffected by begomoviruses and its whitefly vector. An erratum to this article can be found at     

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.     

Genotyping field strains of African swine fever virus by partial p72 gene characterisation

Summary.  A PCR-based sequencing method was developed which permits detection and characterization of African swine fever virus (ASFV) variants within 5 and 48 h, respectively, of receipt of a clinical specimen. Amplification of a 478 bp fragment corresponding to the C-terminal end of the p72 gene, confirms virus presence with genetic characterization being achieved by nucleotide sequence determination and phylogenetic analysis. The method was applied to 55 viruses including those representative of the major ASF lineages identified previously by restriction fragment length polymorphism (RFLP) analysis. Results confirmed that the p72 genotyping method identifies the same major viral groupings. Characterization of additional viruses of diverse geographical, species and temporal origin using the PCR-based method indicated the presence of ten major ASF genotypes on the African continent, the largest of which comprised a group of genetically homogeneous viruses recovered from outbreaks in Europe, South America, the Caribbean and West Africa (the ESAC-WA genotype). In contrast, viruses from southern and East African countries were heterogeneous, with multiple genotypes being present within individual countries. This study provides a rapid and accurate means of determining the genotype of field and outbreak strains of ASF and is therefore useful for molecular epidemiological clarification of ASF. Received July 17, 2002; accepted October 30, 2002     

Molecular interaction between two cassava geminiviruses exhibiting cross-protection

There are increasing reports of geminivirus mixed infections of field plant hosts. These mixed infections have been suggested to result in recombinations, emergence of new viruses and new disease epidemics. We previously reported the occurrence of mixed infection between African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) resulting in severe symptoms in cassava fields in Cameroon. Here, we show that reassortment of DNA-A and DNA-B components of ACMV and EACMCV does not form viable recombinants. However, in the presence of both components of either virus, the DNA-A component of the other virus replicated and spread in the absence of its DNA-B component. This result suggests that failure of ACMV and EACMCV to form viable recombinants is due to the inability of each DNA-A component to trans-replicate the heterologous DNA-B component. This study also shows that ACMV DNA-A induces a resistance to ACMV and EACMCV as indicated by absence or late symptom development. Moreover, this resistance enabled plants to recover from severe symptoms caused by EACMCV in Nicotiana benthamiana, suggesting that the resistance induced is not specific to ACMV and is consistent with the phenomenon of cross-protection between related viruses.     

Methods of surveying the incidence and severity of cassava mosaic disease and whitefly vector populations on cassava in Africa: a review

Field surveys in many cassava growing areas of Africa have assessed the incidence and severity of cassava mosaic disease (CMD), populations of the whitefly vector (Bemisia tabaci), and the distribution of cassava mosaic begomoviruses (CMBs). The methods employed differ greatly between countries and attempts at standardization were made in recent CMD surveys in East and Central Africa, notably in the systemwide Whitefly IPM Project, which provides a paradigm for future work on CMBs and whiteflies on cassava in Africa and also elsewhere. However, there is a need for greater standardization so as to assess the continued expansion of the current CMD pandemic in eastern Africa. Standardized methods will facilitate the collection of reliable data, which can be used to predict future disease spread, develop appropriate management strategies and compare disease development between seasons and locations. In this review, the methods used and the problems encountered during such surveys are discussed and recommendations made on future procedure.