East African Cassava Mosaic Cameroon Virus Research Articles

Gradual Emergence of East African cassava mosaic Cameroon virus in Cassava Farms in Côte d’Ivoire

Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are endemic threats to cassava production, causing significant yield losses. They are caused respectively by begomoviruses and ipomoviruses that are transmitted by whiteflies and infected cuttings. This study aimed to monitor and characterize viruses responsible for these diseases in order to fill existing gaps in understanding their epidemiology in Côte d’Ivoire. Field surveys were conducted in 2016, 2017, and 2020, and no CBSD symptoms were observed. However, an increase in CMD incidence was noted from 45.95% in 2016 to 51.37% in 2020, while CMD severity remained moderate over the years with a mean value of 2.29. The number of whiteflies was relatively low and decreased over the years. Molecular diagnostics carried out on cassava leaf samples allowed the detection of East African cassava mosaic Cameroon virus (EACMCMV) that occurs in single as well as in mixed infection with African cassava mosaic virus (ACMV). Single EACMCMV infection, which was detected only in three agroecological zones of eastern Côte d’Ivoire in 2016, spread throughout the country in 2017 and became more widespread in 2020 with a preponderance in central and southern zones, whereas ACMV and EACMCMV coinfection has spread to the entire zones. Phylogenetic analysis of the viral isolates showed that they are closely related to those from Burkina Faso, Ghana, and Nigeria. This changing population of cassava virus species constitutes a serious threat to cassava cultivation. Understanding the evolutionary dynamics of these viruses could help in adopting better disease management strategies to control the disease.

Genome segment ratios change during whitefly transmission of two bipartite cassava mosaic begomoviruses

Cassava mosaic disease is caused by a complex of whitefly-transmitted begomoviruses, which often occur in co-infections. These viruses have bipartite genomes consisting of DNA-A and DNA-B that are encapsidated into separate virions. Individual viruses exist in plants and whitefly vectors as populations comprising both genome segments, which can occur at different frequencies. Both segments are required for infection, and must be transmitted for virus spread to occur. Cassava plants infected with African cassava mosaic virus (ACMV) and/or East African cassava mosaic Cameroon virus (EACMCV), in which the ratios of DNA-A:DNA-B titers differed between plants, were used to examine how titers of the segments in a plant relate to their respective probabilities of acquisition by whiteflies and to the titers of each segment acquired and subsequently transmitted by whiteflies. The probabilities of acquiring each segment of ACMV did not reflect their relative titers in the source plant but they did for EACMCV. However, for both viruses, DNA-A:DNA-B ratios acquired by whiteflies differed from those in the source plant and the ratios transmitted by the whitefly did not differ from one – the ratio at which the highest probability of transmitting both segments is expected.

Cassava begomovirus species diversity changes during plant vegetative cycles.

Cassava is a root crop important for global food security and the third biggest source of calories on the African continent. Cassava production is threatened by Cassava mosaic disease (CMD), which is caused by a complex of single-stranded DNA viruses (family: Geminiviridae, genus: Begomovirus) that are transmitted by the sweet potato whitefly (Bemisia tabaci). Understanding the dynamics of different cassava mosaic begomovirus (CMB) species through time is important for contextualizing disease trends. Cassava plants with CMD symptoms were sampled in Lake Victoria and coastal regions of Kenya before transfer to a greenhouse setting and regular propagation. The field-collected and greenhouse samples were sequenced using Illumina short-read sequencing and analyzed on the Galaxy platform. In the field-collected samples, African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Kenya virus (EACMKV), and East African cassava mosaic virus-Uganda variant (EACMV-Ug) were detected in samples from the Lake Victoria region, while EACMV and East African mosaic Zanzibar virus (EACMZV) were found in the coastal region. Many of the field-collected samples had mixed infections of EACMV and another begomovirus. After 3 years of regrowth in the greenhouse, only EACMV-like viruses were detected in all samples. The results suggest that in these samples, EACMV becomes the dominant virus through vegetative propagation in a greenhouse. This differed from whitefly transmission results. Cassava plants were inoculated with ACMV and another EACMV-like virus, East African cassava mosaic Cameroon virus (EACMCV). Only ACMV was transmitted by whiteflies from these plants to recipient plants, as indicated by sequencing reads and copy number data. These results suggest that whitefly transmission and vegetative transmission lead to different outcomes for ACMV and EACMV-like viruses.

Occurrence of cassava mosaic begomoviruses in national cassava germplasm preserved in two agro‐ecological zones of Ivory Coast

AbstractCassava production in Ivory Coast is hampered by cassava mosaic disease (CMD), which is caused by several begomovirus species. To increase cassava production and conserve genetic resources, the Centre National de Recherche Agronomique (CNRA) maintains a germplasm collection of 610 cassava accessions of various origins, which is kept in open fields in Bouaké and Man. We carried out an epidemiological assessment of the collection and a molecular characterization of the viruses infecting the CNRA cassava germplasm. The field in Man was less affected by CMD (incidence 49.78%, severity 2.23, whitefly infection 2.23%), despite the abundant whitefly populations (1.34 per plant), compared to Bouaké (incidence 74.54%, severity 2.92, whitefly infection 8.04% and whitefly population of 0.34 per plant). However, the predominant mode of CMD transmission was through infected cassava cuttings at both sites. PCR analysis showed that the East African cassava mosaic Cameroon virus (EACMCMV) was always found in co‐infection with African cassava mosaic virus (ACMV), which also occurred in single infection. Co‐infections of EACMCMV and ACMV were more prevalent in Bouaké (52.64%) than Man (13.87%). Eleven complete genome sequences of cassava mosaic begomoviruses infecting the CNRA germplasm were obtained, and phylogenetic analysis showed that they are closely related to ACMV and EACMCMV isolates from Ghana, Burkina Faso and Nigeria. The results of this study will assist breeders to screen accurately for CMD resistance in progeny resulting from parental lines, enabling the strategic distribution of new clean cassava planting materials to reduce the impact of CMD.

Phenotypic screening and molecular characterization of cassava mosaic disease resistance in Côte d'Ivoire cassava germplasm

Cassava is the staple food crop for hundreds of millions of people in Africa. In Côte d'Ivoire, it is a main source of calories for over 26 million people. However, cassava mosaic disease (CMD), caused by cassava mosaic geminiviruses (CMGs) threaten its production. The development, adoption, and use of CMD resistant varieties remain a key CMD management strategy. Therefore, 610 accessions from the Côte d'Ivoire national cassava germplasm maintained by Centre National de Recherche Agronomique (CNRA) research stations in the cities of Man and Bouaké were characterized to assess their resistance to CMD. We found 72 accessions which were symptomless at both sites. Thirty five out of these 72 accessions were infected by African cassava mosaic virus (ACMV) without any CMD symptoms and 37 were virus free. None of the 72 symptomless accessions were infected by East African cassava mosaic Cameroon virus (EACMCMV). The remaining 538 accessions were CMD infected and displayed clear CMD symptoms. The promising 72 accessions (10 locals and 62 improved) were then tested for the presence of resistance genes, CMD1, CMD2 or CMD3. Except for 2 accessions for which no resistance gene was found, the remaining 70 accessions contained one or more resistance genes. Genotyping of the 69 symptomless accessions revealed that 56 were unique genotypes and 13 were potential duplicates. Accessions were structured in three groups with the variabilities at the individual's level. These findings reveal the existence of potential tolerant/resistant cassava accessions in CNRA's germplasm, which can contribute to CMD control and the increase of cassava production in Côte d'Ivoire.

Epidemiological assessment of cassava mosaic disease in Burkina Faso.

Surveys were conducted in 2016 and 2017 across the main cassava‐growing regions of Burkina Faso to assess the status of cassava mosaic disease (CMD) and to determine the virus strains causing the disease, using field observation and phylogenetic analysis. CMD incidence varied between regions and across years but was lowest in Hauts‐Bassins (6.0%, 2016 and 5.4%, 2017) and highest in Centre‐Sud (18.5%, 2016) and in Boucle du Mouhoun (51.7%, 2017). The lowest CMD severity was found in Est region (2.0) for both years and the highest in Sud‐Ouest region (3.3, 2016) and Centre‐Sud region (2.8, 2017). The CMD infection was primarily associated with contaminated cuttings in all regions except in Hauts‐Bassins, where whitefly‐borne infection was higher than cuttings‐borne infection in 2016. PCR screening of 687 samples coupled with sequence analysis revealed the presence of African cassava mosaic‐like (ACMV‐like) viruses and East African cassava mosaic‐like (EACMV‐like) viruses as single infections at 79.5% and 1.1%, respectively. Co‐infections of ACMV‐like and EACMV‐like viruses were detected in 19.4% of the tested samples. In addition, 86.7% of the samples positive for EACMV‐like virus were found to be positive for East African cassava mosaic Cameroon virus (EACMCMV). Phylogenetic analysis revealed the segregation of cassava mosaic geminiviruses (CMGs) from Burkina Faso into three clades specific to ACMV, African cassava mosaic Burkina Faso virus (ACMBFV), and EACMCMV, confirming the presence of these viruses. The results of this study show that EACMCMV occurrence may be more prevalent in Burkina Faso than previously thought.

Acibenzolar-S-methyl induces resistance against cassava mosaic geminiviruses in Nicotiana benthamiana and their vector Bemisia tabaci in cassava (Manihot esculenta)

Cassava mosaic disease (CMD), caused by cassava mosaic geminiviruses (CMGs), is a major constraint to the cassava crop in Africa and southeastern Asia. Here, we investigated the ability of acibenzolar-S-methyl (ASM), a functional analog of salicylic acid (SA), to trigger systemic acquired resistance (SAR) against two CMGs, namely African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in Nicotiana benthamiana. ASM treatment delayed the time to first viral symptoms appearance, reduced virus infection rate, and attenuated symptoms. Furthermore, ASM caused an enhanced recovery from symptoms of both viruses and inhibited plant death observed in N. benthamiana plants infected by EACMCV. This study further showed that ASM induced resistance to the whitefly Bemisia tabaci (Gennadius), the vector of CMGs, in cassava. Notably, we observed that ASM treatment affected adult whitefly feeding preference and oviposition in cassava under both choice and no-choice conditions. A significant reduction in whitefly adult, egg, and nymph populations was observed irrespective of ASM treatment. The results of this study show that ASM has the potential to control both CMGs and their whitefly vector which is an important first step toward managing whitefly and cassava viruses.

Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation.

Cassava mosaic disease (CMD) represents a serious threat to cassava, a major root crop for more than 300 million Africans. CMD is caused by single-stranded DNA begomoviruses that evolve rapidly, making it challenging to develop durable disease resistance. In addition to the evolutionary forces of mutation, recombination and reassortment, factors such as climate, agriculture practices and the presence of DNA satellites may impact viral diversity. To gain insight into the factors that alter and shape viral diversity in planta, we used high-throughput sequencing to characterize the accumulation of nucleotide diversity after inoculation of infectious clones corresponding to African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in the susceptible cassava landrace Kibandameno. We found that vegetative propagation had a significant effect on viral nucleotide diversity, while temperature and a satellite DNA did not have measurable impacts in our study. EACMCV diversity increased linearly with the number of vegetative propagation passages, while ACMV diversity increased for a time and then decreased in later passages. We observed a substitution bias toward C→T and G→A for mutations in the viral genomes consistent with field isolates. Non-coding regions excluding the promoter regions of genes showed the highest levels of nucleotide diversity for each genome component. Changes in the 5′ intergenic region of DNA-A resembled the sequence of the cognate DNA-B sequence. The majority of nucleotide changes in coding regions were non-synonymous, most with predicted deleterious effects on protein structure, indicative of relaxed selection pressure over six vegetative passages. Overall, these results underscore the importance of knowing how cropping practices affect viral evolution and disease progression.

Deeply Sequenced Infectious Clones of Key Cassava Begomovirus Isolates from Cameroon.

We deeply sequenced two pairs of widely used infectious clones (4 plasmids) of the bipartite begomoviruses African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV). The ACMV clones were quite divergent from published sequences. Raw reads, consensus plasmid sequences, and the infectious clones themselves are all publicly available.

South West and North Central Nigeria: Assessment of cassava mosaic disease and field status of African cassava mosaic virus and East African cassava mosaic virus.

Cassava mosaic disease (CMD), caused by cassava mosaic begomoviruses (CMBs), is a major threat to cassava production in Nigeria. The predominant CMBs in Nigeria are African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV) and East African cassava mosaic Cameroon virus (EACMCV), which are transmitted through infected stem cuttings and whitefly vectors. This study was conducted in 2015 and 2017 to assess the epidemiology of CMD and the current distribution of CMBs in cassava farms in South West (SW) and North Central (NC) Nigeria. A survey of cassava farms was undertaken, and samples representative of disease symptoms were collected and assessed using molecular techniques. A total of 184 and 328 cassava farms were sampled in 2015 and 2017, respectively. CMD incidence for both regions surveyed was 43.80 and 12.25% in 2015 and 2017, respectively. Fields in SW recorded a higher incidence rate in 2015 (SW: 45.11%, NC: 42.47%), while the reverse occurred in 2017 (SW: 10.90%, NC: 14.01%). Overall, the CMD incidence in Benue State (NC) was significantly higher than other locations surveyed in both years. CMD symptom severity and mean whitefly population were higher in SW Nigeria in the two survey years. ACMV was widespread across both zones, occurring in 79.1% (453/613) and 54.8% (386/704) of cassava leaf samples analysed in 2015 and 2017, respectively. EACMV was detected in only 6.0% (37/613) and 4.7% (33/704) of all cassava leaf samples analysed in 2015 and 2017, respectively. Overall, a higher proportion of infected samples were found in NC in both 2015 (NC: 85.2%, SW: 75.4%) and 2017 (NC: 73.6%, SW: 45.2%). Detection using strain‐specific primers revealed that 97% of EACMV positive samples were indeed infected by the EACMCV strain of the virus. As previously reported, samples with mixed infections showed a higher symptom severity than samples with single ACMV or EACMV infections. This study provides an update to the distribution of CMBs in SW and NC Nigeria and will be useful in development of monitoring and management strategies for the disease in both regions.

The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection.

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

Two Novel DNAs That Enhance Symptoms and Overcome CMD2 Resistance to Cassava Mosaic Disease.

ABSTRACTCassava mosaic begomoviruses (CMBs) cause cassava mosaic disease (CMD) across Africa and the Indian subcontinent. Like all members of the geminivirus family, CMBs have small, circular single-stranded DNA genomes. We report here the discovery of two novel DNA sequences, designated SEGS-1 and SEGS-2 (for sequences enhancing geminivirus symptoms), that enhance symptoms and break resistance to CMD. The SEGS are characterized by GC-rich regions and the absence of long open reading frames. Both SEGS enhanced CMD symptoms in cassava (Manihot esculenta Crantz) when coinoculated with African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), or East African cassava mosaic virus-Uganda (EACMV-UG). SEGS-1 also overcame resistance of a cassava landrace carrying the CMD2 resistance locus when coinoculated with EACMV-UG. Episomal forms of both SEGS were detected in CMB-infected cassava but not in healthy cassava. SEGS-2 episomes were also found in virions and whiteflies. SEGS-1 has no homology to geminiviruses or their associated satellites, but the cassava genome contains a sequence that is 99% identical to full-length SEGS-1. The cassava genome also includes three sequences with 84 to 89% identity to SEGS-2 that together encompass all of SEGS-2 except for a 52-bp region, which includes the episomal junction and a 26-bp sequence related to alphasatellite replication origins. These results suggest that SEGS-1 is derived from the cassava genome and facilitates CMB infection as an integrated copy and/or an episome, while SEGS-2 was originally from the cassava genome but now is encapsidated into virions and transmitted as an episome by whiteflies.IMPORTANCE Cassava is a major crop in the developing world, with its production in Africa being second only to maize. CMD is one of the most important diseases of cassava and a serious constraint to production across Africa. CMD2 is a major CMD resistance locus that has been deployed in many cassava cultivars through large-scale breeding programs. In recent years, severe, atypical CMD symptoms have been observed occasionally on resistant cultivars, some of which carry the CMD2 locus, in African fields. In this report, we identified and characterized two DNA sequences, SEGS-1 and SEGS-2, which produce similar symptoms when coinoculated with cassava mosaic begomoviruses onto a susceptible cultivar or a CMD2-resistant landrace. The ability of SEGS-1 to overcome CMD2 resistance and the transmission of SEGS-2 by whiteflies has major implications for the long-term durability of CMD2 resistance and underscore the need for alternative sources of resistance in cassava.

Molecular epidemiology of cassava mosaic disease in Madagascar

Cassava is the staple food for hundreds of millions of people in Africa but its cultivation is seriously constrained by cassava mosaic disease (CMD) in Madagascar, and in Africa in general. This study identified the cassava mosaic geminiviruses (CMGs) involved in CMD in Madagascar and their associated epidemiological characteristics from countrywide surveys. Molecular characterization of CMGs in Madagascar revealed an unprecedented diversity and co‐occurrence of six viruses: African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Kenya virus (EACMKV), East African cassava mosaic virus (EACMV), South African cassava mosaic virus (SACMV) and the recently described Cassava mosaic Madagascar virus (CMMGV). Distinct geographical distributions were observed for the six viruses. While ACMV was more prevalent in the central highlands, EACMV and EACMKV were prevalent in lowlands and coastal regions. Both EACMCV and SACMV occurred in almost all the localities visited. PCR diagnosis revealed that mixed infection (up to four co‐infected viruses) occurred in 21% of the samples and were associated with higher symptom severity scores. Pairwise comparisons of virus associations showed that EACMCV was found in mixed infections more often than expected while ACMV and SACMV were mostly found in single infections. A greater abundance of whiteflies was observed in lowland and coastal areas. Nevertheless, infected cuttings remain the primary source of CMD propagation (95%) in Madagascar.

English

Cassava mosaic disease (CMD), caused by an array of is the most economically important viral disease of cassava in sub-Saharan Africa. The most frequently reported in West Africa are African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV). In this study, 42 cassava leaves and 30 symptomatic weeds belonging to the Asteraceae, Cucurbitaceae and Leguminosae families were collected from backyard gardens in Edo, Ondo, Anambra, and Delta States in 2009. Deoxyribonucleic acid (DNA) extracts from these leaves were tested for ACMV and EACMCV in a multiplex polymerase chain reaction (PCR) assay. The PCR primers used were designed to amplify the replicase regions of DNA-A components of both viruses. Most of the cassava plants within the survey area were either symptomless or showed mild symptoms. ACMV was detected in 16% of cassava leaves from Edo State but not in any of the cassava leaves from the other three states. One weed sample each from Edo State (5.56%) and Ondo State (10%) were also positive for ACMV. EACMCV was not detected in any of the samples tested. The low virus occurrence observed from PCR results and the observed low incidence of the CMD characteristic mosaic symptoms on cassava leaves in the states sampled may be attributed to the use of CMD resistant or tolerant cassava varieties, and may be a result of the massive distribution of virus resistant cassava cuttings to these States by the International Institute of Tropical Agriculture (IITA).   Key words: African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), multiplex polymerase chain reaction (PCR).

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.

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.

First report of the East African cassava mosaic virus‐Uganda (EACMV‐UG) infecting cassava (Manihot esculenta) in Cameroon

Cassava mosaic disease (CMD) is a major constraint of cassava ( Manihot esculenta ) in Cameroon. Previous studies confirmed the occurrence of African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV) and East …

Characterization of the cassava geminivirus transcription activation protein putative nuclear localization signal

The geminivirus transcription activation protein (TrAP) localizes to the nucleus and contains a putative nuclear localization signal (NLS) ( 28PRRRR 32) on the N-terminus. The role of individual residues of this putative NLS on nuclear localization and symptom induction was investigated using TrAP of East African cassava mosaic Cameroon virus (EACMCV). Subcellular localization was conducted using the green fluorescent protein (GFP). Results showed that the proline residue at position 28 (Pro-28) is essential for nuclear localization whereas individually, none of the four contiguous arginines is necessary for nuclear targeting. The role of each of the five NLS amino acid residues on TrAP-mediated disease phenotype and gene silencing suppression was investigated by expressing these mutants in Nicotiana benthamiana from the PVX vector and under the control of the Cauliflower mosaic virus 35S promoter. Results showed that all five residues of the NLS play a role on disease phenotype production in N. benthamiana plants. Furthermore, each of the NLS residues appeared to be required for suppression of VIGS but appeared not to be required for the ability of TrAP to transactivate transcription and interact with adenosine kinase (ADK).

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.

Multiplex PCR for the detection of African cassava mosaic virus and East African cassava mosaic Cameroon virus in cassava

A multiplex PCR was developed for simultaneous detection of African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in cassava affected with cassava mosaic disease (CMD). One set of three primers consisting of an upstream primer common for both viruses and two down stream virus-specific primers were designed for simultaneous amplification of 368 base pair (bp) and 650 bp DNA fragments specific to the replicase gene of ACMV and EACMCV, respectively. Similarly, a second set of three primers were designed for simultaneous amplification of 540 bp and 655 bp fragments specific to the coat protein gene of EACMCV and ACMV, respectively. Primers that can amplify a 171 bp fragment of the large subunit of ribulose bisphosphate carboxylase oxygenase L were included as an internal control in these assays to determine the reliability of multiplex PCR. A simplified, cost-effective and rapid sample preparation method was adapted in place of the conventional plant DNA extraction procedure for multiplex PCR detection of ACMV and EACMCV. The method was validated using CMD-infected cassava samples obtained from farmers’ fields in Nigeria. The multiplex PCR is useful for reliable assessment of the prevalence of CMBs in epidemiological studies and for crop improvement and phytosanitary programs in African countries.