Estimation of Genetic Diversity and Number of Unique Genotypes of Cassava Germplasm from Burkina Faso Using Microsatellite Markers.

Genetic diversity is very important in crop improvement. This study was carried out to assess the genetic diversity and the number of unique multilocus genotypes (MLGs) in a cassava collection in Burkina Faso. To achieve this objective, 130 cassava accessions were genotyped using 32 simple sequence repeat (SSR) markers. The results revealed that among these markers, twelve (12) were highly informative, with polymorphic information content (PIC) values greater than 0.50; twelve (12) were moderately informative, with PIC values ranging between 0.25 and 0.50; and eight (8) were not very informative, with PIC values lower than 0.25. A moderate level of genetic diversity was found for the population, indicated by the average expected heterozygosity (0.45) and the observed heterozygosity (0.48). About 83.8% of unique multilocus genotypes were found in the cassava collection, indicating that SSR markers seem to be most appropriate for MLG identification. Population structure analysis based on hierarchical clustering identified two subpopulations and the Bayesian approach suggested five clusters. Additionally, discriminant analysis of principal components (DAPC) separated the cassava accessions into 13 subpopulations. A comparison of these results and those of a previous study using single nucleotide polymorphisms (SNP) suggests that each type of marker can be used to assess the genetic structure of cassava grown in Burkina Faso.

Cassava mosaic disease and its whitefly vector in Cameroon: Incidence, severity and whitefly numbers from field surveys.

Cassava plays a key role in the food security and economy of Cameroon, but its production is constrained by cassava mosaic disease (CMD). However, comprehensive surveys of CMD in Cameroon have been lacking. This study aimed at evaluating the current status of CMD and its whitefly vector. Field surveys were conducted in 2020 using a sampling, diagnostics and data storage protocol that has been harmonized across 10 West and Central African countries for ease of comparison. Thirty plants per field were assessed for CMD severity, whitefly abundance and source of infection. Surveys were conducted in 343 fields and confirmed the presence of CMD in all 10 regions of Cameroon. Among the 10,057 assessed plants, 33.07% were deemed healthy (asymptomatic). At the field level, only 6.7% fields were found to be healthy. The mean CMD incidence across the country was 66.93%, and the mean severity score was 2.28. The main mode of infection was likely through contaminated cuttings. The mean whitefly count per plant was 5.78. This study is the first countrywide survey of CMD in Cameroon and provides insights that can be useful for improving the country's CMD intervention and management strategies.

Cassava mosaic disease (CMD) in Benin: Incidence, severity and its whitefly abundance from field surveys in 2020.

Cassava mosaic disease (CMD) is the main threat to cassava (Manihot esculenta Crantz) production in Benin. This study was conducted to assess CMD incidence, disease severity, and adult whitefly (Bemisia tabaci) populations in 11 regions of Benin. A total of 180 cassava fields across the country were assessed during June-December 2020 following the harmonized protocol of the Central and West African Virus Epidemiology program. Based on symptoms observation, CMD was present in all surveyed fields in Ouémé and Alibori regions. The highest disease incidence levels were observed in Malanville (100%), Kpomassè (86.67%), Kandi and Zagnanado (both 81.67%), Ségbanan (80%), and Avrankou (76.67%) districts. The highest mean severity scores were in Couffo (3.68), Mono (3.63), and Atlantique (3.33) regions, while the lowest was in Alibori (2.37). Adult whitefly populations (mean number/plant) were highest in Couffo (15.88) and Mono (13.00) regions and lowest in Donga (0.06). Significant relationships were found between CMD severity and whitefly abundance (P = 0.0010) but there was no significant relationship between whitefly numbers and CMD incidence (P = 0.0577). These findings indicate that CMD has expanded its range across Benin. They also provide a basis for designing a response strategy for the control of cassava virus diseases such as CMD.

Survey dataset on the epidemiological assessment of cassava mosaic disease in South West and North Central regions of Nigeria reveals predominance of single viral infection.

The dataset presented here was collected during field surveys conducted in 2015 and 2017, to determine the distribution of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) across 12 Nigerian states and the Federal Capital Territory (FCT), Abuja. In each state, cassava farms were systematically sampled at 10 km intervals except in locations with sparse distribution of cassava farms. In each farm, 30 cassava plants were visually assessed for presence or absence of cassava mosaic disease (CMD) foliar symptoms along two diagonals. Whitefly population was assessed by counting the number of whiteflies on the top five leaves of each sampled plant. Then an average of 4 cassava leaf samples were collected from each farm, and screened for ACMV and EACMV infections using polymerase chain reaction. The dataset includes CMD incidence, symptom severity and the relative abundance of whiteflies in each field as well as laboratory results that show the distribution of ACMV and EACMV across the regions surveyed.

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.

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.

Characterization of virus species associated with sweetpotato virus diseases in Burkina Faso.

Sweetpotato (Ipomoea batatas) production in sub-Saharan Africa is severely affected by viral diseases caused by several interacting viruses, including sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), and sweet potato leaf curl virus (SPLCV). However, the aetiology of viral symptoms on sweetpotato is rarely established in most countries in Africa. Here, we aimed to investigate and characterize the incidence of sweetpotato viruses in Burkina Faso. We performed a countrywide survey in 18 districts of Burkina Faso and collected 600 plants, with and without symptoms, from 80 fields. Viral strains were identified using nitrocellulose membrane-ELISA, PCR, and reverse transcription-PCR. Three scions from each of 50 selected plants with symptoms were grafted to healthy Ipomoea setosa and then serological and molecular tests were performed on the 150 recorded samples. Three viruses were detected: 24% of samples were positive for SPFMV, 18% for SPLCV, and 2% for SPCSV. Across all diagnostic tests, 40% of all plant samples were virus-negative. Coinfections were found in 16% of samples. Partial sequences were obtained, including 13 that matched SPFMV, one that matched SPLCV, and one that matched SPCSV. All identified SPFMV isolates belonged to either phylogroup B or A-II. The SPCSV-positive isolates had 98% gene sequence homology with SPCSV-West Africa for the coat protein. Begomovirus-positive isolates clustered with SPLCV-United States. This first study of sweetpotato viral diseases in Burkina Faso indicates widespread occurrence and suggests a need for further epidemiological investigations, breeding programmes focused on virus-resistant varieties, and improved farming practices to control disease spread.

Response of cassava cultivars to African cassava mosaic virus infection across a range of inoculum doses and plant ages.

Cassava production in Africa is constrained by cassava mosaic disease (CMD) that is caused by the Cassava mosaic virus (CMV). The aim of this study was to evaluate the responses of a range of commonly cultivated West African cassava cultivars to varying inoculum doses of African cassava mosaic virus (ACMV). We grafted 10 cultivars of cassava plants with different inoculum doses of CMV (namely two, four, or six CMD-infected buds) when the experimental plants were 8, 10, or 12 weeks old, using non-inoculated plants as controls. Three cultivars showed disease symptoms when grafted with two buds, and four cultivars showed disease symptoms when grafted with four or six buds. Most cultivars became symptomatic six weeks after inoculation, but one ('TMS92/0326') was symptomatic two weeks after inoculation, and two ('Ntollo' and 'Excel') were symptomatic after four weeks. Root weight tended to be lower in the six-bud than in the two-bud dose, and disease severity varied with plant age at inoculation. These results indicate that the level of CMD resistance in cassava cultivars varies with inoculum dose and timing of infection. This will allow appropriate cultivars to be deployed in each production zone of Africa in accordance with the prevalence of CMD.

Evaluation of resistance to cassava mosaic disease in selected African cassava cultivars using combined molecular and greenhouse grafting tools.

Cassava mosaic disease (CMD) threatens cassava (Manihot esculenta) production in Africa. A total of 24 selected cultivars were screened against CMD using combined molecular and greenhouse grafting tools. Disease severity was recorded for 10 weeks after inoculation and the molecular markers associated with CMD2 were detected by PCR. CMD severity significantly differed (P < 0.0001) among cultivars. Twelve cultivars were morphologically resistant and eight of these possessed CMD2 and four did not. These results suggest that there are several CMD-resistant cassava cultivars that could be recommended for on-farm production and for conservation and breeding programs.

Impact of Cultivated Hosts on the Recombination of Cucumber Mosaic Virus.

Cucumber mosaic virus (CMV) is one of the most successful viruses known, infecting over 1,200 species of plants. Like other single-stranded RNA viruses, CMV is known to have a high potential for population diversity due to error-prone replication and short generation times. Recombination is also a mechanism that allows viruses to adapt to new hosts. Host genes have been identified that impact the recombination of RNA viruses by using single-cell yeast systems. To determine the impact that the natural plant host has on virus recombination, we used a high-recombination-frequency strain of CMV, LS-CMV, which belongs to subgroup II, in three different cultivated hosts: Capsicum annuum cv. Marengo (pepper), Nicotiana tabacum cv. Xanthi nc (tobacco), and Cucurbita pepo cv. Black Beauty (zucchini). The recombination frequency was calculated by using an RNA 3 reporter carrying restriction enzyme sites created by introducing silent mutations. Our results show that the recombination frequency of LS-CMV is correlated with the infected host. The recombination events in pepper were 1.8-fold higher than those in tobacco and 5-fold higher than those in zucchini. Furthermore, we observed the generation of defective RNAs in inoculated pepper plants, but not in tobacco or zucchini. These results indicate that the host is involved in both intra- and intermolecular recombination events and that hosts like pepper could foster more rapid evolution of the virus. In addition, we report for the first time the production of defective RNAs in a CMV subgroup II isolate.IMPORTANCE Recombination is an important mechanism used by viruses for their diversification and to adapt to diverse hosts. Understanding the host role in the mechanisms of evolution is important for virus disease management and controlling the emergence of new strains. This study shows the impact that cultivated hosts are playing in the evolution of CMV. Furthermore, our results and previous studies show how some specific hosts could be an ideal environment for the emergence of new viral strains.

Survey of farmers' knowledge of cassava mosaic disease and their preferences for cassava cultivars in three agro-ecological zones in Benin.

BACKGROUND: Cassava is an important crop in Africa that is widely cultivated for its starchy tuberous root, which constitutes a major source of dietary carbohydrates. Cassava mosaic disease (CMD) is the most devastating disease affecting cassava in Africa and causes enormous losses in yield. In Benin, specifically, cultivars resistant to CMD are not commonly planted, and even when CMD is observed in fields, farmers do not implement control measures, presumably because they lack proper knowledge and training. Our study aimed to evaluate farmers' knowledge of CMD to determine whether there is consistency between farmers' criteria for selecting cassava cultivars and the currently CMD-recommended cassava varieties. METHODS: We conducted structured interviews with 369 farmers in 20% of townships in each of three agro-ecological zones in Benin between November 2015 and February 2016. Farmers were selected randomly in each household, and their fields were assessed for CMD incidence and severity. RESULTS: All farmers surveyed, representing a broad demographic pool with regard to education level, age group, and years of experience in cassava production, successfully recognized CMD symptoms in photos, but most (98.60%) said they did not know the causes and vectors of the disease. Most farmers (93.51%) reported that they obtain planting material from neighboring fields or their own fields. In total, 52 unique cultivars were identified, of which 3 (5.76%) were preferred based on their yield and precocity and 3 (5.76%) were preferred based on taste or ability for transformation. The assessment of disease incidence and severity showed that the areas most affected by CMD were Comè Township (37.77% of fields affected) and agro-ecological zone VIII (26.33%). CONCLUSION: Farmers already know how to recognize the symptoms of CMD and could implement control measures against it if they are trained by researchers. Across all surveyed areas, we identified six preferred cultivars based on the four most commonly stated preference criteria (precocity, yield, gari, and taste. Our results suggest that farmers will be more likely to use CMD-resistant cultivars and clean plant material if the plants meet their existing preference criteria. We suggest that CMD-resistant cultivars will be embraced only if the recommended cultivars are strategically aligned with the characteristics desirable to the cassava farmers in each region.

Mutation and recombination frequencies reveal a biological contrast within strains of Cucumber mosaic virus.

UNLABELLED: Recent in planta studies have shown that strains Fny and LS of Cucumber mosaic virus (CMV) display differential genetic diversities, Fny and LS having higher and lower mutation frequencies, respectively (J. S. Pita and M. J. Roossinck, J Virol 87:790­797, 2012 http://dx.doi.org/10.1128/JVI.01891-12). In this article, we show that these virus strains have differential recombination frequencies as well. However, the high-diversity Fny strain is a low-recombination virus, whereas the very-low-diversity LS strain is instead a high-recombination virus. Unlike the mutation frequency that was determined by both RNAs 1 and 2, the control elements of recombination frequency reside predominantly within RNA 2, specifically within the 2a gene. IMPORTANCE: Recombination is an important mechanism in virus evolution that can lead to increased or decreased variation and is a major player in virus speciation events that can lead to emerging viruses. Although viral genomes show very frequent evidence of recombination, details of the mechanism involved in these events are still poorly understood. We show here that the reciprocal effects of high mutation frequency and low recombination frequency (and vice versa) involve the RNA-dependent RNA polymerase of the virus, and we speculate that these evolutionary events are related to differences in processivity for two strains of the same virus.

Fixation of emerging interviral recombinants in cucumber mosaic virus populations.

Interstrain recombinants were observed in the progenies of the Cucumber mosaic virus (CMV) reassortant L(1)L(2)F(3) containing RNAs 1 and 2 from LS-CMV and RNA 3 from Fny-CMV. We characterized these recombinants, and we found that their fixation was controlled by the nature of the replicating RNAs 1 and 2. We demonstrate that the 2b gene partially affects this fixation process, but only in the context of homologous RNAs 1 and 2.

Mapping viral functional domains for genetic diversity in plants.

Cucumber mosaic virus (CMV) comprises numerous isolates with various levels of in-host diversity. Subgroup-distinctive features of the Fny and LS strains provided us with a platform to genetically map the viral control elements for genetic variation in planta. We found that both RNAs 1 and 2 controlled levels of genetic diversity, and further fine mapping revealed that the control elements of mutation frequency reside within the first 596 amino acids (aa) of RNA 1. The 2a/2b overlapping region of the 2a protein also contributed to control of viral genetic variation. Furthermore, the 3' nontranslated region (NTR) of RNA 3 constituted a hot spot of polymorphism, where the majority of fixed mutations found in the population were clustered. The 2b gene of CMV, a viral suppressor of gene silencing, controls the abundance of the fixed mutants in the viral population via a host-dependent mechanism.

Environment determines fidelity for an RNA virus replicase.

The rate of insertion and deletion mutations of the replicase of Cucumber mosaic virus (CMV) was determined in planta by using a parasitic satellite RNA (satRNA) as a reporter. We found that the CMV replicase had different fidelity in different environments, with important implications in viral disease evolution. Insertions were very rare events, irrespective of the region of the satRNA genome assayed and independent of the hosts tested. On the other hand, deletion events were more frequent but were restricted to a highly structured region of the reporter. Deletion mutation rates were different for the two hosts tested, although the mutation distribution was not influenced by the hosts. Moreover, hot spots with high mutation rates were identified on the satRNA genome.

Differential roles of AC2 and AC4 of cassava geminiviruses in mediating synergism and suppression of posttranscriptional gene silencing.

Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-[CM]) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by approximately 8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by approximately 8-fold. An Agrobacterium-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed.