Cassava Mosaic Geminiviruses Research Articles

Reduction of cassava mosaic geminiviruses from infected stem cuttings using salicylic acid, hydrogen peroxide and hot water treatment

AbstractCassava mosaic disease (CMD) causes significant losses in cassava production in Africa. The disease is caused by several cassava mosaic geminiviruses (CMGs) and spread through the use of infected plant materials. The infected plants remain infected throughout their vegetative lifecycle as the disease cannot be controlled by standard plant protection measures. Therefore, it is important to develop inexpensive field‐based methods that can be easily adopted by small‐scale farmers to sanitize geminiviruses‐infected stem cuttings used as planting material. This study aimed at eliminating CMGs from infected cassava stem cuttings by use of salicylic acid (SA), hydrogen peroxide (H2O2) and hot water (HW) treatments. Eight varieties of CMD‐infected stem cuttings were pretreated with SA (1.25, 2.5 and 5 mM), H2O2 (0.5%, 1.0% and 1.5%) or hot water (50 and 55°C) at different exposure times before establishment in the glasshouse for evaluation of subsequent plant growth and geminivirus detection. Based on the cassava varieties used for the different treatments, treatment with hot water at 50°C for 5 min and 1.0% H2O2 for 12 h were the most effective in eliminating CMGs with an efficiency of 81.7% and 77.8%, respectively. Salicylic acid at a concentration of 5 mM for 6 h eliminated the viruses in 65.1% of the cuttings. The methods tested herein have the potential for producing planting materials with significantly reduced CMD risk for smallholder farmers and the cassava industry to meet their increasing demand. It could also contribute to the global exchange of germplasm for conservation and breeding programs.

A new method for the analysis of access period experiments, illustrated with whitefly-borne cassava mosaic begomovirus.

Reports of low transmission efficiency, of a cassava mosaic begomovirus (CMB) in Bemisia tabaci whitefly, diminished the perceived importance of whitefly in CMB epidemics. Studies indicating synergies between B. tabaci and CMB prompt a reconsideration of this assessment. In this paper, we analysed the retention period and infectiousness of CMB-carrying B. tabaci as well as B. tabaci susceptibility to CMB. We assessed the role of low laboratory insect survival in historic reports of a 9d virus retention period. To do this, we introduced Bayesian analyses to an important class of experiment in plant pathology. We were unable to reject a null hypothesis of life-long CMB retention when we accounted for low insect survival. Our analysis confirmed low insect survival, with insects surviving on average for around three days of transfers from the original infected plant to subsequent test plants. Use of the new analysis to account for insect death may lead to re-calibration of retention periods for other important insect-borne plant pathogens. In addition, we showed that B. tabaci susceptibility to CMB is substantially higher than previously thought. We also introduced a technique for high resolution analysis of retention period, showing that B. tabaci infectiousness with CMB was increasing over the first five days of infection.

Cassava mosaic disease in South and Southeast Asia: current status and prospects

Cassava is one of the most important food and industrial crops in Asia, Africa, and South America. Cassava mosaic disease (CMD), caused by cassava mosaic geminivirus, is one of the major constraints to cassava cultivation. In Asia, the disease is caused by the Indian cassava mosaic virus and Sri Lankan cassava mosaic virus. Phytosanitation, vector control, breeding, and genetic engineering strategies have been widely adopted to manage the disease. This study provides a comprehensive review of the disease spread, the development of diagnostic methods, and various approaches employed for the management of CMD in South and Southeast Asia.

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.

Mutations in DNA polymerase δ subunit 1 co-segregate with CMD2-type resistance to Cassava Mosaic Geminiviruses

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses.

Evaluation of Ten Cassava Varieties for Resistance to Cassava Mosaic Disease in Burkina Faso

The whitefly-transmitted cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) is the most important disease threatening the production of cassava (Manihot esculenta Crantz). Among the main measures for controlling CMD, the use of resistant varieties seems to be among the best methods. This study was conducted in 2017/2018 cassava growing season at three locations to evaluate the level of resistance to CMD of 7 elite cassava varieties widely used by farmers and 3 local cassava varieties cultivated in Burkina Faso. Both morphological and molecular markers were used to screen these varieties against CMGs infection. Morphological markers revealed 8 varieties as highly resistant (TMS 91/02312, TMS 92/0067, TMS 92/0325, TMS 92/0427, TMS 4(2)1425, TMS 94/0270, TMS 30572 and Boborola) whilst the two others (Nouhao and Santidougou) were resistant. The molecular markers linked to CMD1 and CMD2 genes were detected in all varieties. The molecular marker associated with CMD3 gene was detected only in the 8 highly resistant varieties. However, whitefly number per plant and disease pressure were low during this study. It was, therefore, concluded that a better assessment of resistance of cassava varieties to CMD in Burkina Faso could be obtained by combining agro-inoculation and molecular screening.

Cassava Brown Streak Disease Response and Association With Agronomic Traits in Elite Nigerian Cassava Cultivars.

Cassava mosaic geminiviruses (CMGs) and cassava brown streak viruses (CBSVs) cause the highest yield losses in cassava production in Africa. In particular, cassava brown streak disease (CBSD) is and continues to be a significant constraint to optimal cassava production in Eastern and Southern Africa. While CBSD has not been reported in West Africa, its recent rapid spread and damage to cassava productivity in Eastern, and Southern Africa is alarming. The aim of this study was to evaluate Nigerian cassava genotypes in order to determine their responses to CBSD, in the event that it invades Nigeria, the world’s largest cassava producer. The study gathered information on whether useful CBSD resistance alleles are present in the elite Nigerian cassava accessions. A total of 1,980 full-sib cassava seedlings from 106 families were assessed in the field at the seedling stage for a year. A subset of 569 clones were selected and assessed for another year at the clonal stage in Namulonge, central Uganda, a known hotspot for CBSD screening. Results indicated that foliar and root incidences and severities varied significantly (p ≤ 0.01, p ≤ 0.001) except for CBSD foliar incidence at 6 months (CBSD6i). Highest and lowest plot-based heritability estimates for CBSD were registered for CBSD root severity (CBSDrs) (0.71) and CBSD6i (0.5). Positive and highly significant correlations were noted between CBSD root incidence (CBSDri) and CBSDrs (r = 0.90***). Significant positive correlations were also noted between CBSD foliar severity at 3 months (CBSD3s) and CBSD foliar incidence at 6 months (CBSD6i) (r = 0.77***), CBSD3s and CBSDrs (r = 0.35***). Fresh root weight (FreshRW) negatively correlated with CBSDri and CBSDrs, respectively (r = −0.21*** and r = −0.22***). Similarly, CBSD3s correlated negatively with cassava mosaic disease severity at 3 (CMD3s) and 6 months (CMD6s), respectively (r = −0.25*** and r = −0.21***). Fifteen clones were selected using a non-weighted summation selection index for further screening. In conclusion, results revealed that the elite Nigerian accessions exhibited significant susceptibility to CBSD within 2 years of evaluation period. It is expected that this information will aid future breeding decisions for the improvement of CBSD resistance among the Nigerian cassava varieties.

Contribution of historical herbarium small RNAs to the reconstruction of a cassava mosaic geminivirus evolutionary history

Emerging viral diseases of plants are recognised as a growing threat to global food security. However, little is known about the evolutionary processes and ecological factors underlying the emergence and success of viruses that have caused past epidemics. With technological advances in the field of ancient genomics, it is now possible to sequence historical genomes to provide a better understanding of viral plant disease emergence and pathogen evolutionary history. In this context, herbarium specimens represent a valuable source of dated and preserved material. We report here the first historical genome of a crop pathogen DNA virus, a 90-year-old African cassava mosaic virus (ACMV), reconstructed from small RNA sequences bearing hallmarks of small interfering RNAs. Relative to tip-calibrated dating inferences using only modern data, those performed with the historical genome yielded both molecular evolution rate estimates that were significantly lower, and lineage divergence times that were significantly older. Crucially, divergence times estimated without the historical genome appeared in discordance with both historical disease reports and the existence of the historical genome itself. In conclusion, our study reports an updated time-frame for the history and evolution of ACMV and illustrates how the study of crop viral diseases could benefit from natural history collections.

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.

Characterization of Sirnas Derived from Two Species of Cassava-Infecting Geminiviruses in Nicotiana Benthamiana

The infection dynamics of cassava mosaic geminiviruses was determined in Nicotiana benthamiana. Following inoculation, symptoms were observed at 5 and 10 dpi for African cassava mosaic virus (ACMV-CM) and the Kenyan strain K201 of East African cassava mosaic virus (EACMV KE2 [K201]). Northern and Southern blots indicated higher accumulation of ACMV-CM at 10 dpi compared with EACMV KE2 (K201) correlating with more severe symptoms at this time point. However, unlike for EACMV KE2 (K201), symptom remission characterized by appearance of non-asymptomatic leaves was observed after 20 dpi in plants infected with ACMV-CM. Deep sequencing identified between 969,015 and 1,307,689 total reads of which >80% mapped to the host genome. For both virus species, 22 nt virus derived sRNAs (vsRNAs) reads were the most abundant (34% - 53%) followed by 21 nt (27% - 27%) and 24 nt (9 – 23%) vsRNA reads. EACMV KE2 (K201) vsRNAs were proportionally higher (12.31% and 4.51%) than those derived from ACMV-CM (5.70% and 0.45%) for DNA A and DNA B respectively. vsRNAs were covered the entire virus genome with dense accumulation at regions where the viral ORFs overlapped. Key words; RNA interference, Cassava mosaic disease, Geminiviruses DOI: 10.7176/JNSR/10-8-06 Publication date: April 30 th 2020

Haplotype-resolved genomes of geminivirus-resistant and geminivirus-susceptible African cassava cultivars

BackgroundCassava is an important food crop in tropical and sub-tropical regions worldwide. In Africa, cassava production is widely affected by cassava mosaic disease (CMD), which is caused by the African cassava mosaic geminivirus that is transmitted by whiteflies. Cassava breeders often use a single locus, CMD2, for introducing CMD resistance into susceptible cultivars. The CMD2 locus has been genetically mapped to a 10-Mbp region, but its organization and genes as well as their functions are unknown.ResultsWe report haplotype-resolved de novo assemblies and annotations of the genomes for the African cassava cultivar TME (tropical Manihot esculenta), which is the origin of CMD2, and the CMD-susceptible cultivar 60444. The assemblies provide phased haplotype information for over 80% of the genomes. Haplotype comparison identified novel features previously hidden in collapsed and fragmented cassava genomes, including thousands of allelic variants, inter-haplotype diversity in coding regions, and patterns of diversification through allele-specific expression. Reconstruction of the CMD2 locus revealed a highly complex region with nearly identical gene sets but limited microsynteny between the two cultivars.ConclusionsThe genome maps of the CMD2 locus in both 60444 and TME3, together with the newly annotated genes, will help the identification of the causal genetic basis of CMD2 resistance to geminiviruses. Our de novo cassava genome assemblies will also facilitate genetic mapping approaches to narrow the large CMD2 region to a few candidate genes for better informed strategies to develop robust geminivirus resistance in susceptible cassava cultivars.

Genetic diversity of Bemisia tabaci species colonizing cassava in Central African Republic characterized by analysis of cytochrome c oxidase subunit I.

After 2007, upsurges of whiteflies on cassava plants and high incidences of cassava diseases were observed in Central African Republic. This recent upsurge in the abundance of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) was directly linked to serious damage to cassava crops resulting from spread of whitefly-borne cassava mosaic geminiviruses (CMGs). There is currently very little information describing whitefly populations on cassava and associated crops in Central African Republic. The current study aimed to address this gap, and to determine whether the increasing damage associated with B. tabaci whiteflies was the consequence of a new invasion, or an upsurge of a local population. The molecular genetic identification and phylogenetic relationships of 898 B. tabaci adult individuals collected from representative locations (54) throughout CAR were determined based on their mitochondrial cytochrome oxidase I sequences (mtCOI). Field and ecological data were also collected from each site, including whitefly abundance, CMD incidence, host plants colonized by B. tabaci and agro-ecological zone. Phylogenetic analysis of the whitefly mtCOI sequences indicated that SSA1 (-SG1, -SG2), SSA3, MED, MEAM1 and Indian Ocean (IO) putative species occur in CAR. One specific haplotype of SSA1-SG1 (SSA1-SG1-P18F5) predominated on most cassava plants and at the majority of sites. This haplotype was identical to the SSA1-SG1 Mukono8-4 (KM377961) haplotype that was recorded from Uganda but that also occurs widely in CMD pandemic-affected areas of East Africa. These results suggest that the SSA1-SG1-P18F5 haplotype occurring in CAR represents a recent invasive population, and that it is the likely cause of the increased spread and severity of CMD in CAR. Furthermore, the high mtDNA sequence diversity observed for SSA1 and its broad presence on all sites and host plants sampled suggest that this genetic group was the dominant resident species even before the arrival of this new invasive haplotype.

Interactions Between Cassava Mosaic Geminiviruses and Their Vector, Bemisia tabaci (Hemiptera: Aleyrodidae).

The sweetpotato whitefly, Bemisia tabaci (Gennadius) is the vector of the cassava mosaic geminiviruses (CMGs) that cause cassava mosaic disease (CMD). Synergistic interactions between B. tabaci and CMGs have been hypothesized as a cause of whitefly "super-abundance," which has been a key factor behind the spread of the severe CMD pandemic through East and Central Africa. The current study investigated this hypothesis by conducting experiments with CMD-susceptible cassava varieties infected with different CMGs in both the north-western Lake Zone region (pandemic affected) and the eastern Coast Zone where CMD is less severe. Male and female pairs of B. tabaci were placed in clip cages for 48 h on plants of three cassava varieties at each of the two locations. There were significantly more eggs laid on CMG-infected than on CMG-free plants in the Lake Zone, whereas in Coast Zone, there were no significant differences. There were no significant differences in proportions, mortality, and development duration of immature stages of B. tabaci among virus states and cassava variety in the two locations. The overall number of eggs was significantly higher with longer development duration of the immature stages in the Lake than in the Coast Zone, whereas mortality was significantly higher in the Coast than in the Lake Zone. Based on these results, it is concluded that there was no net positive synergistic interaction between CMGs and B. tabaci for either lowland coastal or mid-altitude inland populations. Consequently, other factors seem more likely to be the cause of the "super-abundance," and require further investigation.

The Search for Resistance to Cassava Mosaic Geminiviruses: How Much We Have Accomplished, and What Lies Ahead.

The cassava mosaic disease (CMD), which occurs in all cassava growing regions of Africa and the Indian subcontinent, is caused by cassava mosaic geminiviruses (CMGs). CMGs are considered to be the most damaging vector-borne plant pathogens. So far, the most successful approach used to control these viruses has been the transfer of a polygenic recessive resistance locus, designated CMD1, from wild cassava to cassava cultivars. Further progress in harnessing natural resistance to contain CMGs has come from the discovery of the dominant monogenic resistance locus, CMD2, in some West African cassava cultivars. CMD2 has been combined with CMD1 through genetic crosses. Because of the limitations of the cassava breeding approach, especially with regard to time required to produce a variety and the loss of preferred agronomic attributes, efforts have been directed toward the deployment of genetic engineering approaches. Most of these approaches have been centered on RNA silencing strategies, developed mainly in the model plant Nicotiana benthamiana. Early RNA silencing platforms assessed for CMG resistance have been use of viral genes for co-suppression, antisense suppression or for hairpin RNAs-mediated gene silencing. Here, progress and challenges in the deployment of these approaches in the control of CMGs are discussed. Novel functional genomics approaches with potential to overcome some of the drawbacks of the current strategies are also discussed.

Differential response of cassava genotypes to infection by cassava mosaic geminiviruses

Mitigation of cassava mosaic disease (CMD) focuses on the introgression of resistance imparted by the polygenic recessive (CMD1), dominant monogenic (CMD2) and CMD3 loci. The mechanism(s) of resistance they impart, however, remain unknown. Two CMD susceptible and nine CMD resistant cassava genotypes were inoculated by microparticle bombardment with infectious clones of African cassava mosaic virus Cameroon strain (ACMV-CM) and the Kenyan strain K201 of East African cassava mosaic virus (EACMV KE2 [K201]). Genotypes carrying the CMD1 (TMS 30572), CMD2 (TME 3, TME 204 and Oko-iyawo) and CMD3 (TMS 97/0505) resistance mechanisms showed high levels of resistance to ACMV-CM, with viral DNA undetectable by PCR beyond 7days post inoculation (dpi). In contrast, all genotypes initially developed severe CMD symptoms and accumulated high virus titers after inoculation with EACMV KE2 (K201). Resistant genotypes recovered to become asymptomatic by 65dpi with no detectable virus in newly formed leaves. Genotype TMS 97/2205 showed highest resistance to EACMV KE2 (K201) with <30% of inoculated plants developing symptoms followed by complete recovery by 35dpi. Deep sequencing of small RNAs confirmed production of 21–24 nt virus derived small RNAs (vsRNA) that mapped to cover the entire ACMV-CM and EACMV KE2 (K201) viral genomes in both polarities, with hotspots seen within gene coding regions. In resistant genotypes, total vsRNAs were most abundant at 20 and 35dpi but reduced significantly upon recovery from CMD. In contrast, CMD susceptible genotypes displayed abundant vsRNAs throughout the experimental period. The percentage of vsRNAs reads ranked by class size were 21nt (45%), 22 nt (28%) and 24 nt (18%) in all genotypes studied. The number of vsRNA reads directly correlated with virus titer and CMD symptoms.

Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus.

The major threat for cassava cultivation on the Indian subcontinent is cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses which are bipartite begomoviruses with DNA A and DNA B components. Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV) cause CMD in India. Two isolates of SLCMV infected the cassava cultivar Sengutchi in the fields near Malappuram and Thiruvananthapuram cities of Kerala State, India. The Malappuram isolate was persistent when maintained in the Madurai Kamaraj University (MKU, Madurai, Tamil Nadu, India) greenhouse, whereas the Thiruvananthapuram isolate did not persist. The recovered cassava plants with the non-persistent SLCMV, which were maintained vegetative in quarantine in the University of Basel (Basel, Switzerland) greenhouse, displayed re-emergence of CMD after a six-month period. Interestingly, these plants did not carry SLCMV but carried ICMV. It is interpreted that the field-collected, SLCMV-infected cassava plants were co-infected with low levels of ICMV. The loss of SLCMV in recovered cassava plants, under greenhouse conditions, then facilitated the re-emergence of ICMV. The partial dimer clones of the persistent and non-persistent isolates of SLCMV and the re-emerged isolate of ICMV were infective in Nicotiana benthamiana upon agroinoculation. Studies on pseudo-recombination between SLCMV and ICMV in N. benthamiana provided evidence for trans-replication of ICMV DNA B by SLCMV DNA A.

The rolling circle amplification and next generation sequencing approaches reveal genome wide diversity of Kenyan cassava mosaic geminivirus

Rolling circle amplification is a simple approach of enriching populations of single-stranded DNA plant begomovirus genomes (genus, Begomovirus; family, Geminiviridae). This is an innovative approach that utilizes the robustness of the bacteriophage phi29 DNA polymerase used in circle amplification, together with deep sequencing using Illumina Miseq and bioinformatics to assess population diversity of begomoviruses in naturally infected cassava. The approach is suitable for detecting rare members in a population in begomoviral populations in situation where mixtures of isolates, strains, and multiple species occur. The main objectives were to increase the sensitivity of detection of next generation sequencing by enriching it using rolling circle amplification then determination of the diversity of the cassava mosaic geminivirus. This was done by total nucleic acids isolated from sy mptomatic, field cassava infected plants, then using rolling circle amplification to multiply the less abundant viral sequences. Enriched and non-enriched virus-libraries were subjected to deep sequencing using Illumina Miseq. Using bioinformatic CLC Genomics 5.5.1 software programs the quality assessment of reads and contig assembly of viral sequences. This was done through de novo and reference-guided assembly. The identity and diversities of the begomoviral sequences were compared with sequences in Sanger sequencing of viral components deposited in the NCBI Gene Bank. In this study we have demonstrated that RCA increases the chances of detecting the virus by approximately 10 to 1000 fold and wide genome diversity of cassava mosaic geminivirus in various cassava growing zones in Kenya were detected. In conclusion, this approach described herein is simple and will enhance the exploration of begomovirus diversities from cassava infected plants, irrespective of their viral abundance. This will make it possible for routine screening of field samples as the cost of deep sequencing NGS is decreasing and the advances of bioinformatic software development become enhanced. This is the first report of the RCA-Illumina-NGS approach to explore cassava infected with begomoviruses under field conditions and their diversities. Key words : Illumina Miseq sequencing, geminivirus, ssDNA viruses, viral sequence enrichment, de novo genome assembly, rolling cycle amplification (RCA).

English

Rolling circle amplification is a simple approach of enriching populations of single-stranded DNA plant begomovirus genomes (genus, Begomovirus; family, Geminiviridae). This is an innovative approach that utilizes the robustness of the bacteriophage phi29 DNA polymerase used in circle amplification, together with deep sequencing using Illumina Miseq and bioinformatics to assess population diversity of begomoviruses in naturally infected cassava. The approach is suitable for detecting rare members in a population in begomoviral populations in situation where mixtures of isolates, strains, and multiple species occur. The main objectives were to increase the sensitivity of detection of next generation sequencing by enriching it using rolling circle amplification then determination of the diversity of the cassava mosaic geminivirus. This was done by total nucleic acids isolated from symptomatic, field cassava infected plants, then using rolling circle amplification to multiply the less abundant viral sequences. Enriched and non-enriched virus-libraries were subjected to deep sequencing using Illumina Miseq. Using bioinformatic CLC Genomics 5.5.1 software programs the quality assessment of reads and contig assembly of viral sequences. This was done through de novo and reference-guided assembly. The identity and diversities of the begomoviral sequences were compared with sequences in Sanger sequencing of viral components deposited in the NCBI Gene Bank. In this study we have demonstrated that RCA increases the chances of detecting the virus by approximately 10 to 1000 fold and wide genome diversity of cassava mosaic geminivirus in various cassava growing zones in Kenya were detected. In conclusion, this approach described herein is simple and will enhance the exploration of begomovirus diversities from cassava infected plants, irrespective of their viral abundance. This will make it possible for routine screening of field samples as the cost of deep sequencing NGS is decreasing and the advances of bioinformatic software development become enhanced. This is the first report of the RCA-Illumina-NGS approach to explore cassava infected with begomoviruses under field conditions and their diversities. Key words: Illumina Miseq sequencing, geminivirus, ssDNA viruses, viral sequence enrichment, de novo genome assembly, rolling cycle amplification (RCA).

Divergent evolutionary and epidemiological dynamics of cassava mosaic geminiviruses in Madagascar.

BackgroundCassava mosaic disease (CMD) in Madagascar is caused by a complex of at least six African cassava mosaic geminivirus (CMG) species. This provides a rare opportunity for a comparative study of the evolutionary and epidemiological dynamics of distinct pathogenic crop-infecting viral species that coexist within the same environment. The genetic and spatial structure of CMG populations in Madagascar was studied and Bayesian phylogeographic modelling was applied to infer the origins of Madagascan CMG populations within the epidemiological context of related populations situated on mainland Africa and other south western Indian Ocean (SWIO) islands.ResultsThe isolation and analysis of 279 DNA-A and 117 DNA-B sequences revealed the presence in Madagascar of four prevalent CMG species (South African cassava mosaic virus, SACMV; African cassava mosaic virus, ACMV; East African cassava mosaic Kenya virus, EACMKV; and East African cassava mosaic Cameroon virus, EACMCV), and of numerous CMG recombinants that have, to date, only ever been detected on this island. SACMV and ACMV, the two most prevalent viruses, displayed low degrees of genetic diversity and have most likely been introduced to the island only once. By contrast, EACMV-like CMG populations (consisting of East African cassava mosaic virus, EAMCKV, EACMCV and complex recombinants of these) were more diverse, more spatially structured, and displayed evidence of at least three independent introductions from mainland Africa. Although there were no statistically supported virus movement events between Madagascar and the other SWIO islands, at least one mainland African ACMV variant likely originated in Madagascar.ConclusionsOur study highlights both the complexity of CMD in Madagascar, and the distinct evolutionary and spatial dynamics of the different viral species that collectively are associated with this disease. Given that more distinct CMG species and recombinants have been found in Madagascar than any other similarly sized region of the world, the risks of recombinant CMG variants emerging on this island are likely to be higher than elsewhere. Evidence of an epidemiological link between Madagascan and mainland African CMGs suggests that the consequences of such emergence events could reach far beyond the shores of this island.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0749-2) contains supplementary material, which is available to authorized users.