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

Samples 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.     

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

The 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.     

Viruses Associated with Cassava Mosaic Disease in Senegal and Guinea Conakry

A survey in Senegal and Guinea Conakry established the presence and incidence of cassava mosaic virus disease (CMD) in both countries. CMD occurred in all the fields surveyed, although its incidence was higher in Senegal (83%) than in Guinea (64%). Populations of the whitefly vector, Bemisia tabaci, were low in both countries averaging 1.7 adults per shoot in Guinea and 3.2 in Senegal. Most infections were attributed to the use of infected cuttings, 86 and 83% in Senegal and Guinea, respectively, and there was no evidence of rapid current‐season, whitefly‐borne infection at any of the sampled locations. Disease severity was generally low in the two countries and averaged 2.5 in Guinea and 2.3 in Senegal. No plants with unusually severe CMD symptoms characteristic of the CMD pandemic in East and Central Africa were observed. Restriction fragment length polymorphism (RFLP)‐based diagnostics revealed that African cassava mosaic virus (ACMV) is exclusively associated with CMD in both the countries. Neither East African cassava mosaic virus (EACMV), nor the recombinant Uganda variant (EACMV‐UG2) was detected in any sample. These survey data indicate that CMD could be effectively controlled in both countries by phytosanitation, involving the use of CMD‐free planting material and the removal of diseased plants.     

The effect of cassava mosaic geminiviruses on symptom severity, growth and root yield of a cassava mosaic virus disease-susceptible cultivar in Uganda

A study was carried out to assess the effect of different cassava mosaic geminiviruses (CMGs) occurring in Uganda on the growth and yield of the susceptible local cultivar ‘Ebwanateraka’. Plants infected with African cassava mosaic virus (ACMV), ‘mild’ and ‘severe’ strains of East African cassava mosaic virus‐Uganda (EACMV‐UG2) and both ACMV and EACMV‐UG2 were grown in two experiments in Kabula, Lyantonde in western Uganda. The most severe disease developed in plants co‐infected with ACMV and EACMV‐UG2 and in those infected with the ‘severe’ form of EACMV‐UG2 alone; disease was least severe in plants infected with the ‘mild’ strain of EACMV‐UG2. ACMV‐infected plants and those infected with the ‘mild’ strain of EACMV‐UG2 were tallest in the 1999–2000 and 2000–2001 trials, respectively; plants dually infected with ACMV and EACMV‐UG2 were shortest in both trials. Plants infected with ‘mild’ EACMV‐UG2 yielded the largest number and the heaviest tuberous roots followed by ACMV and EACMV‐UG2 ‘severe’, respectively, whilst plants dually infected with ACMV and EACMV‐UG2 yielded the least considering the two trials together. Reduction in tuberous root weight was greatest in plants dually infected with ACMV and EACMV‐UG2, averaging 82%. Losses attributed to ACMV alone, EACMV‐UG2 ‘mild’ and EACMV‐UG2 ‘severe’ were 42%, 12% and 68%, respectively. Fifty percent and 48% of the plants infected with both ACMV and EACMV‐UG2 gave no root yield in 1999–2000 and 2000–2001, respectively. These results indicate that CMGs, whether in single or mixed infections, reduce root yield and numbers of tuberous roots produced and that losses are substantially increased following mixed infection.     

Molecular Variability and Distribution of Cassava Mosaic Begomoviruses in Nigeria

Several begomovirus species and strains causing Cassava mosaic disease (CMD) have been reported from cassava in Africa. In Nigeria, African cassava mosaic virus (ACMV) was the predominant virus in this important crop, and East African cassava mosaic virus (EACMV), first reported from eastern Nigeria in 1999, was also found occasionally. A survey was conducted in 2002 to resolve the diversity of the virus types present in cassava in Nigeria and to further understand the increasing complexity of the viruses contributing to CMD. A total of 234 leaf samples from cassava with conspicuous CMD symptoms were collected in farmers’ fields across different agroecological zones of Nigeria and subjected to polymerase chain reaction (PCR) with type‐specific primers. In addition and, to provide a full characterization of the viruses present, DNA‐A genome components of several viruses and informative genome fragments were sequenced. In Nigeria, ACMV proved to be the dominant virus with 80% of all samples being positive for ACMV. The East African cassava mosaic Cameroon virus (EACMCV) prevalent in Cameroon and Ivory Coast was detected in single infections (2%) and in mixed infections (18%) with ACMV. There was no indication for other virus strains of EACMV present in the country. The EACMCV samples collected showed a high nucleotide sequence identity >98% and resembled the described sequence of a Cameroon isolate (EACMCV‐CM) more than an Ivory Coast isolate, EACMCV‐CM[CI]. Evidence is provided that the EACMCV has reached epidemiological significance in Nigeria.     

Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses

Cassava is infected by numerous geminiviruses in Africa and India that cause devastating losses to poor farmers. We here describe the molecular diversity of seven representative cassava mosaic geminiviruses (CMGs) infecting cassava from multiple locations in Tanzania. We report for the first time the presence of two isolates in East Africa: (EACMCV-[TZ1] and EACMCV-[TZ7]) of the species East African cassava mosaic Cameroon virus, originally described in West Africa. The complete nucleotide sequence of EACMCV-[TZ1] DNA-A and DNA-B components shared a high overall sequence identity to EACMCV-[CM] components (92% and 84%). The EACMCV-[TZ1] and -[TZ7] genomic components have recombinations in the same genome regions reported in EACMCV-[CM], but they also have additional recombinations in both components. Evidence from sequence analysis suggests that the two strains have the same ancient origin and are not recent introductions. EACMCV-[TZ1] occurred widely in the southern part of the country. Four other CMG isolates were identified: two were close to the EACMV-Kenya strain (named EACMV-[KE/TZT] and EACMV-[KE/TZM] with 96% sequence identity); one isolate, TZ10, had 98% homology to EACMV-UG2Svr and was named EACMV-UG2 [TZ10]; and finally one isolate was 95% identical to EACMV-[TZ] and named EACMV-[TZ/YV]. One isolate of African cassava mosaic virus with 97% sequence identity with other isolates of ACMV was named ACMV-[TZ]. It represents the first ACMV isolate from Tanzania to be sequenced. The molecular variability of CMGs was also evaluated using partial B component nucleotide sequences of 13 EACMV isolates from Tanzania. Using the sequences of all CMGs currently available, we have shown the presence of a number of putative recombination fragments that are more prominent in all components of EACMV than in ACMV. This new knowledge about the molecular CMG diversity in East Africa, and in Tanzania in particular, has led us to hypothesize about the probable importance of this part of Africa as a source of diversity and evolutionary change both during the early stages of the relationship between CMGs and cassava and in more recent times. The existence of multiple CMG isolates with high DNA genome diversity in Tanzania and the molecular forces behind this diversity pose a threat to cassava production throughout the African continent.     

Role of a novel type of double infection in the geminivirus-induced epidemic of severe cassava mosaic in Uganda

To study the cause of the current epidemic of severe mosaic in Ugandan cassava, PCR analysis was used to detect and identify African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and the recently reported recombinant geminivirus (UgV), which is derived from ACMV and EACMV, in leaf extracts from cassava plants grown from cuttings in the glasshouse at Dundee. The cuttings were collected from plants showing symptoms of different severities and growing at different sites in Uganda inside, at the periphery of, and outside, the area affected by the epidemic. ACMV occurred throughout the nine districts sampled but UgV was detected only in the area affected by the epidemic. EACMV was not found in Uganda. Most plants containing ACMV alone expressed mild or moderate mosaic, whereas very severe mosaic developed in most plants containing UgV plus ACMV and a few of those containing UgV only. Very severe mosaic in cassava from southern Sudan was likewise associated with co-infection by UgV and ACMV. The very severe disease was reproduced by graft-inoculating geminivirus-free cassava with UgV plus ACMV; plants inoculated with either UgV or ACMV developed severe or moderate symptoms, respectively. Unlike ACMV, Malawian EACMV did not enhance the severity of symptoms induced by UgV. However, a very severely affected plant from Ukerewe Island, Tanzania, contained ACMV and EACMV but not UgV. UgV attained a much greater concentration in cassava than did ACMV but the opposite occurred in Nicotiana benthamiana. In neither host was total virus antigen concentration affected by co-infection. Factors affecting the genesis, selection and spread of UgV are discussed. The evidence indicates that UgV is probably of relatively recent origin, that such variants do not appear often, and that the current epidemic has resulted from the rapid spread of UgV to infect plants and to invade regions in which ACMV already occurred. The novel type of virus complex so produced, consisting of an interspecific recombinant virus (UgV) and one of its parents (ACMV), typically has even more severe effects than UgV alone.     

Occurrence of three distinct begomoviruses in cassava in Madagascar

The presence of East African cassava mosaic virus in association with cassava mosaic disease in Madagascar has previously been reported. We now describe virus isolates from mosaic‐affected Madagascan cassava with epitope profiles typical of African cassava mosaic virus, and an isolate with a nucleotide sequence similar to that of South African cassava mosaic virus. Thus, three distinct begomoviruses occur in cassava in Madagascar.     

Efficacy of chemotherapy and thermotherapy in elimination of East African cassava mosaic virus from Tanzanian cassava landrace

Cassava mosaic disease is caused by cassava mosaic begomoviruses (CMBs) and can result in crop losses up to 100% in cassava (Manihot esculenta) in Tanzania. We investigated the efficacy of chemotherapy and thermotherapy for elimination of East African cassava mosaic virus (EACMV) of Tanzanian cassava. In vitro plantlets from EACMV‐infected plants obtained from coastal Tanzania were established in the greenhouse. Leaves were sampled from the plants and tested to confirm the presence of EACMV. Plantlets of plants positive for EACMV were initiated in Murashige and Skoog (MS) medium. On the second subculture, they were subjected into chemical treatment in the medium containing salicylic acid (0, 10, 20, 30 and 40 mg/L) and ribavirin (0, 5, 10, 15 and 20 mg/L). In the second experiment, EACMV‐infected plantlets were subjected to temperatures between 35 and 40°C with 28°C as the control. After 42 days of growth, DNA was extracted from plant leaves and PCR amplification was performed using EACMV specific primers. It was found that plant survival decreased with increasing levels of both salicylic acid and ribavirin concentrations. In general, plants treated with salicylic acid exhibited a lower plant survival % than those treated with ribavirin. However, the percentage of virus‐free plants increased with an increase in the concentration of both ribavirin and salicylic acid. The most effective concentrations were 20 mg/L of ribavirin and 30 mg/L of salicylic acid; these resulted in 85.0% and 88.9% virus‐free plantlets, respectively. With regard to thermotherapy, 35°C resulted in 79.5% virus‐free plantlets compared to 69.5% at 40°C. Based on virus elimination, ribavirin at 20 mg/L, salicylic acid 30 mg/L and thermotherapy at 35°C are recommended for production of EACMV free cassava plantlets from infected cassava landraces.     

Exploiting the Combination of Natural and Genetically Engineered Resistance to Cassava Mosaic and Cassava Brown Streak Viruses Impacting Cassava Production in Africa

Cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) are currently two major viral diseases that severely reduce cassava production in large areas of Sub-Saharan Africa. Natural resistance has so far only been reported for CMD in cassava. CBSD is caused by two virus species, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). A sequence of the CBSV coat protein (CP) highly conserved between the two virus species was used to demonstrate that a CBSV-CP hairpin construct sufficed to generate immunity against both viral species in the cassava model cultivar (cv. 60444). Most of the transgenic lines showed high levels of resistance under increasing viral loads using a stringent top-grafting method of inoculation. No viral replication was observed in the resistant transgenic lines and they remained free of typical CBSD root symptoms 7 month post-infection. To generate transgenic cassava lines combining resistance to both CBSD and CMD the hairpin construct was transferred to a CMD-resistant farmer-preferred Nigerian landrace TME 7 (Oko-Iyawo). An adapted protocol allowed the efficient Agrobacterium-based transformation of TME 7 and the regeneration of transgenic lines with high levels of CBSV-CP hairpin-derived small RNAs. All transgenic TME 7 lines were immune to both CBSV and UCBSV infections. Further evaluation of the transgenic TME 7 lines revealed that CBSD resistance was maintained when plants were co-inoculated with East African cassava mosaic virus (EACMV), a geminivirus causing CMD. The innovative combination of natural and engineered virus resistance in farmer-preferred landraces will be particularly important to reducing the increasing impact of cassava viral diseases in Africa.     

Disease resistance characterisation of improved cassava genotypes to cassava mosaic disease at different ecozones

Twenty-two cassava genotypes and eight controls were evaluated in two cropping seasons for resistance to cassava mosaic disease (CMD) at the International Institute of Tropical Agriculture (IITA) fields, located at different ecozones of Nigeria. Disease incidence (DI) and index of symptom severity data were obtained monthly at each location and genotype. Symptomatic leaves were also collected during evaluation at each location, and virus was indexed by amplification in polymerase chain reaction. Significant differences within and across locations were observed in the reactions of cassava genotypes to CMD. DI across cassava genotypes was significantly (p = 0.05) highest in the Ibadan (22.6%), followed by Onne (19.3%). Generally, plants of clones 96/0860, 96/1439, 96/0160, 96/1089A, 96/1632, 96/1613, 96/1708, 96/0191, 96/0249 and 96/1565 had significantly lower values of DI in each location. African cassava mosaic virus in single infection was the predominant causal agent of CMD in IITA experimental fields under study.     

Current characteristics of cassava mosaic disease in postepidemic areas increase the range of possible management options

As a case study to document the current characteristics of cassava mosaic virus disease (CMD) in postepidemic areas, surveys were carried out, in 2003 and 2004, in Siaya District of western Kenya. This was an area affected by a severe CMD pandemic in the late 1990s. Data recorded on cassava varieties were CMD incidence, severity index and number of adult whiteflies. Farmers (174) were interviewed on their understanding of the disease and their knowledge and practice of management interventions. Cassava cultivation was being re‐established, but local landraces predominated. Resistant varieties were present 13% in 2003, and 4% in 2004, of the surveyed fields. Adhiambolera was the most common variety, occurring in 35% and 40% of fields in 2003 and 2004, respectively, and had an average CMD incidence of 82% in 2003 and 73% in 2004. By contrast, the CMD‐resistant variety Migyera had a low mean incidence (28% in 2003). The overall incidence for both years was 71%, consisting of 61% as a result of infection through planting diseased cuttings and 10% as a result of whitefly infection. In 2003, the total incidence was 72% and the average severity 2.7 (severity index), while in 2004 the incidence was 78% and the severity 2.6. There were significant severity variations in each division of the Siaya District during the 2 years except for Karemo and Ukwala. The abundance of whiteflies on the top five leaves of plants was low in 2003 but high in 2004, with means of 1 and 16, respectively, over the same seven divisions in both years, although this variation was thought to be because of seasonal factors. East African cassava mosaic virus‐Uganda was the predominant geminivirus present in every division. Phytosanitation by farmers was minimal, as evidenced by 29% of farmers using a selection of CMD‐free stems for planting and 15% using hand‐roguing for CMD management. Occurrence of more than 25% CMD‐free plants in 2004, moderate CMD severity and limited spread provide a conducive environment for the use of phytosanitation as a CMD control measure that can be immediately used by farmers growing their own cassava varieties.     

Virus content of leaves of cassava infected by African cassava mosaic virus

African cassava mosaic virus (ACMV) was detected in cassava leaves by ELISA. Some normal constituents of cassava leaves interfered with virus detection but leaf extracts of Nicotiana benthamiana did not. The symptom pattern was determined early in the growth of a leaf and subsequently changed little. ACMV was found only in the yellow or yellow green areas of the mosaic pattern. Virus content of the leaves increased with increasing symptom intensity, but decreased with leaf age and ACMV was not detected in mature leaves. Most whiteflies were found on young growing cassava leaves and the number decreased progressively with leaf age. This distribution will aid both the acquisition and inoculation of the virus.     

Genetic diversity and geographic distribution of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) genotypes associated with cassava in East Africa

The genetic variability of whitefly (Bemisia tabaci) species, the vectors of cassava mosaic begomoviruses (CMBs) in cassava growing areas of Kenya, Tanzania, and Uganda, was investigated through comparison of partial sequences of the mitochondria cytochrome oxidase I (mtCOI) DNA in 2010/11. Two distinct species were obtained including sub‐Saharan Africa 1 (SSA1), comprising of two sub‐clades (I and II), and a South West Indian Ocean Islands (SWIO) species. Among the SSA1, sub‐clade I sequences shared a similarity of 97.8–99.7% with the published Uganda 1 genotypes, and diverged by 0.3–2.2%. A pairwise comparison of SSA1 sub‐clade II sequences revealed a similarity of 97.2–99.5% with reference southern Africa genotypes, and diverged by 0.5–2.8%. The SSA1 sub‐clade I whiteflies were widely distributed in East Africa (EA). In comparison, the SSA1 sub‐clade II whiteflies were detected for the first time in the EA region, and occurred predominantly in the coast regions of Kenya, southern and coast Tanzania. They occurred in low abundance in the Lake Victoria Basin of Tanzania and were widespread in all four regions in Uganda. The SWIO species had a sequence similarity of 97.2–97.7% with the published Reunion sequence and diverged by 2.3–2.8%. The SWIO whiteflies occurred in coast Kenya only. The sub‐Saharan Africa 2 whitefly species (Ug2) that was associated with the severe CMD pandemic in Uganda was not detected in our study.     

Transgenic cassava resistance to <Emphasis Type="Italic">African cassava mosaic virus</Emphasis> is enhanced by viral DNA-A bidirectional promoter-derived siRNAs

Expression of double-stranded RNA (dsRNA) homologous to virus sequences can effectively interfere with RNA virus infection in plant cells by triggering RNA silencing. Here we applied this approach against a DNA virus, African cassava mosaic virus (ACMV), in its natural host cassava. Transgenic cassava plants were developed to express small interfering RNAs (siRNA) from a CaMV 35S promoter-controlled, intron-containing dsRNA cognate to the common region-containing bidirectional promoter of ACMV DNA-A. In two of three independent transgenic lines, accelerated plant recovery from ACMV-NOg infection was observed, which correlates with the presence of transgene-derived siRNAs 21–24 nt in length. Overall, cassava mosaic disease symptoms were dramatically attenuated in these two lines and less viral DNA accumulation was detected in their leaves than in those of wild-type plants. In a transient replication assay using leaf disks from the two transgenic lines, strongly reduced accumulation of viral single-stranded DNA was observed. Our study suggests that a natural RNA silencing mechanism targeting DNA viruses through production of virus-derived siRNAs is turned on earlier and more efficiently in transgenic plants expressing dsRNA cognate to the viral promoter and common region.     

A Method for Selection of Tolerance to African Cassava Mosaic Virus

Detopping hastened the appearance of mosaic symptoms in shoots of cassava cuttings infected with African cassava mosaic virus (ACMV). The degree of plant recovery from severe mosaic after each detopping was the basis for evaluation of ACMV tolerance. In field-screening, detopping of cassava shoots infected with ACMV is recommended for selection of tolerance to the virus. One detopping of ACMV-infected shoots was sufficient to analyze tolerance level reliably. Out of 521 lines only one was found tobe tolerant, while 144 lines were found to be moderately tolerant. These included the lines ‘Gimbi MA 235’, (02945S × 3119)S MA 219, and ‘Kadanga Malombe’ previously described as resistant to ACMV.     

Serological detection and antigenic variation of two whitefly-transmitted geminiviruses: tobacco leaf curl and croton yellow vein mosaic viruses

A panel of 25 monoclonal antibodies (MAbs) raised against particles of two heterologous whitefly-transmitted geminiviruses (begomoviruses) was used in triple antibody-sandwich ELISA (TAS-ELISA) to determine the detectability and epitope profiles of 26 Indian isolates of tobacco leaf curl virus (TLCV) and 13 of croton yellow vein mosaic virus (CYVMV). Stock cultures of the two viruses had indistinguishable epitope profiles although they differ in symptomatology and particle stability. Their epitope profiles also strongly resembled those of Indian isolates of bhendi (okra) yellow vein mosaic and Indian cassava mosaic (ICMV) viruses. TLCV isolates from Andhra Pradesh, Gujarat and Karnataka States differed slightly in epitope profile: they reacted with at least eight out of 10 MAbs raised to ICMV but only one to four out of 15 MAbs raised to African cassava mosaic virus (ACMV). Virus isolates serologically indistinguishable from TLCV were detected in symptom-bearing weeds (Acanthospermum hispidum, Ageratum conyzoides, Euphorbia geniculata, Parthenium hysterophorus) found in leaf curl-affected tobacco fields and shown previously to be experimental hosts of TLCV. Indian TLCV isolates had small, consistent differences in epitope profile from Pakistani isolates but large differences from isolates from Burkina Faso, Malawi or Uganda. Isolates from the three African countries reacted with four or five of the ACMV MAbs but only one or two of the ICMV MAbs, and there were small but consistent inter-country differences. CYVMV isolates from three Indian States showed less epitope variation than did Indian isolates of TLCV. TAS-ELISA with MAb SCR 18 was a more sensitive test for detecting Indian TLCV isolates than was double antibody-sandwich ELISA with polyclonal antibodies.     

Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

Background

Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses.

Results

All genes encoded by four distinct begomoviruses (African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct.

Conclusions

Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case.     

Occurrence of whitefly-transmitted geminiviruses in crops in Burkina Faso, and their serological detection and differentiation

Whitefly-transmitted geminiviruses were found to be associated with four diseases of crop plants in Burkina Faso: cassava mosaic, okra leaf curl, tobacco leaf curl and tomato yellow leaf curl. Tomato yellow leaf curl is an economically serious disease, reaching a high incidence in March, following a peak population of the vector whitefly, Bemisia tabaci, in December. Okra leaf curl is also a problem in the small area of okra grown in the dry season but is not important in the main period of okra production in the rainy season. The geminiviruses causing these four diseases, African cassava mosaic (ACMV), okra leaf curl (OLCV), tobacco leaf curl (TobLCV) and tomato yellow leaf curl (TYLCV) viruses, were each detected in field-collected samples by triple antibody sand-wich-ELISA with cross-reacting monoclonal antibodies (MAbs) to ACMV. Epitope profiles obtained by testing each virus isolate with panels of MAbs to ACMV, OLCV and Indian cassava mosaic virus enabled four viruses to be distinguished. ACMV and OLCV had similar but distinguishable profiles. The epitope profile of TobLCV was the same as that of one form of TYLCV (which may be the same virus) and was close to the profile of TYLCV from Sardinia. The other form of TYLCV reacted with several additional MAbs and had an epitope profile close to that of TYLCV from Senegal. Only minor variations within each of these four types of epitope profile were found among geminivirus isolates from Burkina Faso. Sida acuta is a wild host of OLCV.