Cassava Mosaic Geminiviruses Research Articles

The cassava mosaic geminiviruses occurring in Uganda following the 1990s epidemic of severe cassava mosaic disease

SummaryThe cassava mosaic geminiviruses (CMGs) isolated from cassava plants expressing mild and severe symptoms of cassava mosaic disease (CMD) in 2002 in Uganda were investigated using the polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) molecular techniques and DNA sequencing. Two previously described cassava mosaic geminiviruses: African cassava mosaic virus (ACMV) said East African cassava mosaic virus ‐ Uganda variant (EACMV‐UG2) were detected in Uganda. The RFLP technique distinguished two polymorphic variants of ACMV (ACMV‐UG1 and ACMV‐UG2) and three of EACMV‐UG2 (EACMV‐UG2[1], EACMV‐UG2[2] and EACMV‐UG2[3]). ACMV‐UG1 produced the fragments predicted for the published sequences of ACMV‐[KE]/UGMld/ UGSvr, while ACMV‐UG2, which produced the RFLP fragments predicted for the West African ACMV isolates ACMV‐[NG], ACMV‐[CM], ACMV‐[CM/DO2] and ACMV‐[CI], was shown to be ACMV‐UGMld/UGSvr after DNA sequencing. EACMV‐UG2[1] produced the RFLP fragments predicted for the published sequences of EACMV‐UG2/UG2Mld/UG2Svr. However, both EACMV‐UG2[2] and EACMV‐UG2[3], which produced East African cassava mosaic vzras‐[Tanzania]‐like polymorphic fragments with RFLP analysis, were confirmed to be isolates of EACMV‐UG2 after DNA sequencing. Thus, this study emphasises the importance of DNA sequence analysis for the identification of CMG isolates. EACMV‐UG2 was the predominant virus and occurred in all the surveyed regions. It was detected in 73% of the severely and 53% of the mildly diseased plants, while ACMV was less widespread and occurred most frequently in the mildly diseased plants (in 27% of these plants). Mixed infections of ACMV and EACMV‐UG2 were detected in only 18% of the field samples. Unlike previously reported results the mixed infection occurred almost equally in plants exhibiting mild or severe disease symptoms (21% and 16%, respectively). The increasing frequency of mild forms of EACMV‐UG2 together with the continued occurrence of severe forms in the field warrants further studies of virus‐virus and virus‐host interactions.

Geminate structures of African cassava mosaic virus.

Two types of geminate structures were purified from African cassava mosaic geminivirus (ACMV)-infected Nicotiana benthamiana plants and analyzed by electron cryomicroscopy and image reconstruction. After cesium sulfate density gradient centrifugation, they were separated into lighter top (T) and heavier bottom (B) components. T particles comigrated with host proteins, whereas B particles were concentrated in a cesium density typical for complete virions. Both particles were composed of two incomplete icosahedra of 11 capsomers each, but T particles were slightly larger (diameter, 22.5 nm) and less dense in the interior than B particles (diameter, 21.5 nm). T particles were frequently associated with small globules of approximately 14 nm diameter of unknown origin. The overall structure of ACMV, a begomovirus transmitted by whiteflies, was similar to that of Maize streak virus (MSV), a mastrevirus transmitted by leafhoppers, although the vertices of the icosahedra were less pronounced. Models of ACMV coat proteins based on Satellite tobacco necrosis virus support the exposure of parts of the molecule essential for transmission specificity by whiteflies and provide possible structural explanations for the smaller protrusion of the ACMV capsid relative to MSV. The differences of ACMV and MSV virion shapes are discussed with reference to their different animal vectors.

First report of cassava mosaic disease and cassava mosaic geminiviruses in Gabon

Cassava mosaic disease (CMD), arguably Africa's greatest plant protection problem, has been known to occur in central/West Africa for more than 70 years. There is, however, no published record of the occurrence of CMD or the cassava mosaic geminiviruses (CMGs) that cause it from the central African country of Gabon. Significantly, however, the severe Uganda variant of East African cassava mosaic virus (EACMV-UG), associated with the African CMD pandemic, has been recorded from the Republic of Congo (Neuenschwander et al., 2002), bordering Gabon to the west. Cassava fields were examined at 55 sites throughout Gabon in April and July 2003, in order to identify the CMGs associated with CMD and to determine if the pandemic-associated EACMV-UG was present. At each site, 30 plants were examined and assessments made of CMD incidence, symptom severity and the abundance of the whitefly vector Bemisia tabaci. At least two virus-diseased leaf samples were collected from each field for subsequent virus diagnosis. DNA was extracted from these samples on the day of collection using the method of Dellaporta et al. (1983). Virus diagnoses were subsequently made from DNA samples using both specific primer PCR (Zhou et al., 1997) and RFLP analysis involving restriction digestion by EcoRV and MluI of near full-length DNA-A fragments amplified using abutting primer PCR with universal geminivirus primers (Briddon & Markham, 1994). ACMV was the most widely distributed CMG species, occurring at 53 of the 55 sites. EACMV was identified from a single site just south of Tchibanga in southern Gabon. EACMV-UG was detected in samples collected from 12 sites, all of which were in the eastern provinces of Haute-Ogooué and Ogooué-Ivindo. Of the 17 samples infected with EACMV-UG, 16 were mixed infections with ACMV. Symptom severity of plants infected by EACMV-UG (4·1) was significantly greater (χ2 = 44·4, d.f. = 3, P < 0·001) than that of plants infected by ACMV alone (2·8). Additionally, four of the five virus-sampled plants having current season whitefly-borne CMD were dual ACMV + EACMV-UG infections. These data comprise the first published record of CMGs in Gabon. The results also describe an early stage of spread of the EACMV-UG-associated CMD pandemic into eastern Gabon, which now represents the pandemic's westernmost ‘front’. Grant assistance from the Office for US Foreign Disaster Assistance and the technical help of Dr M. Tindo and Mr R. Obonyo are acknowledged.

Field Studies of Cross Protection with Cassava Mosaic Geminiviruses in Uganda

AbstractThe effect of cassava mosaic virus disease (CMD) was compared on plants grown from cuttings that were initially virus‐free at planting and those infected with a mild strain of East African cassava mosaic virus‐Uganda (EACMV‐UG). All initially healthy plants developed CMD symptoms within 5 months of planting (MAP) at both trial sites in Uganda, although spread was more rapid at Kamuli than at Serere. Significantly (P &lt; 0.001) higher symptom severity scores were recorded in initially healthy plants, which had average scores of 3.6 and 3.5 at Kamuli and Serere, respectively, compared with 2.8 for mildly diseased plants at each location. Severity scores of 4 and 5 were more frequent in initially healthy plants, accounting for 77 and 39% of the total infections recorded in comparison with 47 and 11% in mildly diseased plants at Kamuli and Serere, respectively. Mildly diseased plants were significantly taller than initially healthy plants 8 and 12 MAP at both locations. However, the converse was true 4 MAP although differences were significant at Serere but not at Kamuli. Mildly diseased plants yielded significantly more tuberous roots than initially healthy plants at Kamuli but not at Serere. Average total weights of tuberous roots per plant were 2.48 and 1.63 kg for mildly diseased and initially healthy plants at Kamuli and 4.46 and 4.61 kg at Serere, respectively. These results may help to explain the increased prevalence in recent years of mildly diseased plants of local CMD‐susceptible cultivars in eastern Uganda, from where these varieties virtually disappeared following the severe CMD epidemic in the 1990s. The results also provide the first field evidence of a cross protective effect of mild strains of a cassava mosaic geminivirus.

Effects of cassava genotype, climate and the Bemisia tabaci vector population on the development of African cassava mosaic geminivirus (ACMV)

A survey was carried out in the 1996/97 and 1997/98 growing seasons on a field planted in three replicates with five clones of cassava at the International Institute of Tropical Agriculture, Ibadan, located in a transition forest, to determine the effects of cassava genotype and climate on the development of African cassava mosaic geminivirus (ACMV) and changes in the Bemisiatabaci population. Cassava genotype, climate and their interactions have significant (P&lt;0.01) effects on the population of B. tabaci and the development of ACMV. The incidence of ACMV was significantly (P&lt;0.01) higher in clones 81/01635 and 92/0520 than in TMS 30572 and 94/0239, while 91/02327 showed the greatest resistance. A positive correlation between the incidence and severity of ACMV was observed, but this did not correlate with the whitefly population density.

Occurrence of the Uganda variant of East African cassava mosaic virus (EACMV‐Ug) in western Democratic Republic of Congo and the Congo Republic defines the westernmost extent of the CMD pandemic in East/Central Africa

), which causesunusually severe CMD symptoms, was investigated.Cassava leaves with severe CMD symptoms were col-lected in August 1999 from nine fields in Lekana, N’Sahand Abala-Ndolo in the central part of Congo Republic,and leaves with a range of symptoms were collected inDecember 2000 from 30 CMD-affected plants from 12fields in Kinshasa and Bas-Congo Provinces of DRC.Samples from the Congo Republic were preserved inalcohol and tested for the presence of cassava mosaic gemini-viruses at IITA-Ibadan by PCR using nonspecificEACMV primers and specific primers for

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.

Subcellular targeting of the coat protein of African cassava mosaic geminivirus.

The coat protein (CP) of geminiviruses is involved in a number of processes during the life cycle of the virus. The predominant function is encapsidation of single-stranded DNA and formation of the virus particle to protect viral DNA during transmission. The CP of monopartite geminiviruses is absolutely essential for virus movement, whereas CP mutants of bipartite geminiviruses are able to infect some host plants systemically, indicating an involvement of the CP in host specificity. During the life cycle of geminiviruses, the viral DNA enters the nucleus of the infected cell where virus replication, transcription, and encapsidation occur. For systemic infection, the virus moves cell-to-cell from the site of inoculation to vascular tissue and via phloem to other plant tissues. To move, viral DNA has to shuttle in and out of the nucleus and through plasmodesmata. Parts of the bipartite African cassava mosaic virus (ACMV) CP were fused with green fluorescent protein (GFP) or beta-glucuronidase (GUS). CP domains were identified that mediate both nuclear import and export, as well as targeting of CP-fusion proteins to the cell periphery. These results indicate that domains of the CP facilitate several aspects of geminivirus movement, including nuclear import and export and transport of the viral genome to the cell periphery.

Genomic and biological diversity of the African cassava geminiviruses

The virological situation of cassava in Africa is increasing in complexity due to the number and types of viruses isolated from different locations within the continent. Here, we report the complete nucleotide sequences of both A and B components of two geminivirus species infecting cassava in the Ivory Coast and review the current knowledge of the molecular and biological diversity of the African cassava geminiviruses. As a whole, newly obtained sequences are compared with those of the African cassava mosaic geminiviruses identified to date. Results indicate that all isolates of African cassava mosaic virus (ACMV), irrespective of their geographical origin are clustered together with little or no variation in their genomic sequence. On the contrary, the genomes of the East African cassava mosaic virus (EACMV) are more genetically diverse due to the frequent occurrence of recombinations within their two components. Indeed, the EACMV-like viruses vary so much that their classification is becoming problematic. In addition, there is also a large range of phenotypic symptom variation for each of these virus species, irrespective of the location of isolation. Furthermore, it has been shown that ACMV and EACMV can be synergistic in cassava, resulting in a greater DNA accumulation and consequently inducing severe symptoms. For all these reasons, this paper initiates a discussion concerning the species demarcation for cassava geminivirus.

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.

Field experiments in Cameroon on cassava mosaic virus disease and the reversion phenomenon in susceptible and resistant cassava cultivars

Cassava is mainly propagated through stem-cuttings. There is evidence that some cuttings propagated from plants infected by cassava mosaic geminiviruses may produce virus-free plants. Field experiments were conducted under contrasting ecological conditions at two sites in southern Cameroon to study the reversion phenomenon, in particular the extent to which the virus causing cassava mosaic disease (CMD) was absent from cuttings collected from CMD-affected plants. Cuttings were taken from the basal, middle and upper portions of the stems of diseased plants of susceptible and partially resistant cassava cultivars. The proportions of disease-free shoots developing from the different batches of cuttings were recorded to indicate the extent of reversion. This was significantly less frequent in cv. Red Local (susceptible) (5-43%) than in the susceptible cv. Bambui Local (65%) and cv. Improved (70%), which is partially resistant to infection. Reversion was significantly higher with cuttings collected from the youngest portions of the stem compared with older portions and with short cuttings (5, 10 cm) compared with those 20 or 25 cm long. There was a significant negative correlation between symptom severity in different cultivars and the extent of reversion. This was greater at the lowland Mile 17 site, near Buea (ca 400 masl) than at the mid-altitude site at Dschang (ca 1300 masl) where conditions were cooler, suggesting an influence of temperature. In order to assess the effect of CMD in cassava the Disease Index (DI) was developed. The result of DI analyses showed that the DI could be used to identify cultivars on the basis of their ability to revert from CMD and the severity of CMD on the non-reverted plants.

Identification of genes directly and indirectly involved in the insect transmission of African cassava mosaic geminivirus by Bemisia tabaci.

The inability to transmit progeny virus resulting from the cloned components of an isolate of African cassava mosaic virus originating from Kenya (ACMV-K) has been shown to be due to defects in both genomic components. This was achieved by the production of infectious pseudorecombinants between ACMV-K and the cloned components of a whitefly-transmissible ACMV isolate originating from Nigeria (ACMV-NOg). The exchange of gene fragments between ACMV-K and ACMV-NOg has been used to demonstrate that the defects responsible for lack of transmissibility reside on the coat protein and DNA B C1 gene of ACMV-K. The significance of these finding with respect to the present understanding of the function of these gene products are discussed.

Recombination between viral DNA and the transgenic coat protein gene of African cassava mosaic geminivirus.

Nicotiana benthamiana was transformed with three different constructs (pCRA1, pCRA2 and pJC1) containing the coat protein coding sequence of African cassava mosaic virus (ACMV). Transformed plants were inoculated with a coat protein deletion mutant of ACMV that induces mild systemic symptoms in control plants. Several inoculated plants of transgenic lines CRA1/3, CRA1/4, CRA2/1 and CRA2/2 developed severe systemic symptoms typical of ACMV. DNA analysis revealed that, in these plants, recombination had occurred between the mutant viral DNA and the integrated construct DNA, resulting in the production of recombinant virus progeny with 'wild-type' characteristics. No reversion of mutant to 'wild-type' virus was observed in pJC1-transformed plants. Recombinant virus from several transgenic plants was analysed by PCR and parts of DNA A of virus progeny were cloned. Sequence analysis revealed that only a few nucleotides were changed from the published sequence.

Survey of cassava virus diseases in Tanzania

A survey was carried out of African cassava mosaic geminivirus and cassava brown streak virus diseases (ACMD and CBSD) in Tanzania. ACMD occurred throughout the country at low to moderate incidences in the fifteen different regions surveyed (1- 64%). The incidence was generally higher along the coastalplain than in higher altitude areas in the interior. There was a significant correlation between numbers ofadults ofthe whitefly vector, Bemisia tabaci, and incidence of recently infected plants ( R = 0.77, p < 0.01), although most infection was attributable to the use of infected cuttings. CBSD was more restricted in distribution and, where present, incidence was low to moderate (1- 36%). Cassava plantings along the southern border with Mozambique link the highest incidence area on the south-east coast near Mtwara, with the other important area ofCBSD incidence along the shore ofLake Malawi. The effects of climatic parameters on ACMD and CBSD incidence are discussed. The survey data suggest that both diseases could be controlled in Tanzania by the use of phytosanitation which involves the use of disease-free planting material and the removal (roguing) of diseased plants.

Formation of Circular Satellite Tobacco Ringspot Virus RNA in Protoplasts Transiently Expressing the Linear RNA

The most abundant form of the satellite RNA of tobacco ringspot virus (sTRSV RNA) is a linear, unit length molecule of 359 nucleotide residues, designated L−(+)M. A postulated replication scheme for the satellite RNA has as its first, and apparently virus-independent, step the ligation of L−(+)M into the corresponding circular form C−(+)M. We transiently expressed L−(+)M wild type and L−(+)M mutants in tobacco protoplasts using an African cassava mosaic geminivirus vector. Measured extents of C−(+)M accumulation were correlated with computer-predicted folding to suggest wild-type secondary structure elements that might be deleted without reducing ligation. A 127-nucleotide residue mutant L−(+)M was created by replacing, with 7 and 3 residues, respectively, nucleotide residues 53–211 and 268–350, each of which was predicted to form a set of three adjacent imperfect stem-loops in wild-type L−(+)M. The mutant L−(+)M was found to be extensively ligated to C−(+)M in protoplasts and to retain a calculated helix of the wild-type molecule that incorporates the 3′ terminal sequence. A trinucleotide in the 3′ region was mutated so as to disrupt and restore, respectively, the calculated helix, reducing and restoring, respectively, C−(+)M formation. These results suggest that the 3′ stem contributes to the suitability of the small L−(+)M molecules as a substrate for a protoplast RNA ligase and that computed folding of sTRSV RNA may be predictive of sTRSV RNA structurein vivo.

Efficient whitefly transmission of African cassava mosaic geminivirus requires sequences from both genomic components.

Clones of two subgroup III geminiviruses, the common strain of tomato golden mosaic virus (csTGMV) and African cassava mosaic virus originating from Kenya (ACMV-K), were shown to be non-transmissible by whitefiles. Lack of transmissibility of cloned ACMV-K was investigated by exchanging genomic components with a whitefly-transmissible ACMV isolate from Nigeria (ACMV-NOg). Neither pseudorecombinant was transmissible, indicating that defects in both genomic components contributed to the lack of transmissibility. Analysis of the acquisition of the pseudorecombinats by Bemisia tabaci indicated that accumulation of virus within the insect was DNA B dependent. Return of virus to plants was determined by DNA A, although the coat protein was essential for acquisition. Repeated passaging of both the wild strain of ACMV-NOg and the cloned virus led to loss of insect transmissibility of the wild isolate but not the cloned virus. Products encoded on both genomic components are required for transmission of bipartite geminiviruses by B. tabaci.

Host‐associated strains within Ugandan populations of the whitefly Bemisia tabaci (Genn.),(Hom., Aleyrodidae)

Abstract: Studies were undertaken to investigate host relationships of populations of Bemisia tabaci taken from the major crop hosts in Uganda. B. tabaci adults taken from cotton and sweet potato did not colonize cassava. None survived for longer than two days and nymphs emerging from eggs laid on cassava died. Cassava B. tabaci in contrast showed limited colonization on cotton and sweet potato and adults emerging from cotton retained the ability to colonize cassava, confirming the genetic basis of the distinction between ‘strains’ suggested by previous isozyme studies. The implications of these findings with regard to the role of B. tabaci in the epidemiology of African cassava mosaic geminivirus are discussed.

Effective ribozyme delivery in plant cells.

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.

The phenology ofBemisia tabaci(Homoptera: Aleyrodidae) populations on cassava in southern Côte d'Ivoire

AbstractPopulation phenologies of the whitefly,Bemisia tabaci(Gennadius) (Homoptera: Aleyrodidae), in young cassava crops in Côte d'Ivoire, West Africa, are described for three field seasons. Populations of all stages were consistently greatest 6–12 weeks after the crop was planted. The number of adults on plants as well as on attractive and non-attractive sticky traps displayed cycles of buildup and decline each year, the periodicity of these cycles corresponding to the generation time ofB. tabaciunder field conditions. Adult population declines were probably caused by emigration from the crop. Rainfall was negatively correlated with both nymph and adult populations, possibly due to reduced oviposition after rain.B. tabaciis the vector of African cassava mosaic geminiviruses (ACMV) and the observedB. tabacipopulation trends fit well with the pattern of ACMV buildup in the crop.

A geminivirus causing vein yellowing of Ageratum conyzoides in Singapore

A vein‐yellowing disease of Ageratum conyzoides in Singapore was shown to be caused by a geminivirus, here named ageratum yellow vein virus (AYVV), which was transmitted by the whitefly Bemisia tabaci but not by inoculation with sap or through seed. AYVV particles (30 × 20 nm) are serologically related to those of other whitefly‐transmitted geminiviruses, and reacted with some monoclonal antibodies elicited by particles of African cassava mosaic or Indian cassava mosaic geminiviruses. However, the epitope profile of AYVV differed from the profiles of these viruses, and from those of geminiviruses from vein yellowing‐affected A. conyzoides from India and from yellow leaf curl‐affected tomato from either Singapore or India. The results provide further evidence of antigenic differences among geminiviruses that cause similar diseases in the same plant species in different geographical regions.