Ugandan Cassava Brown Streak Virus Research Articles

Assessing the effect of sample storage time on viral detection using a rapid and cost-effective CTAB-based extraction method

BackgroundCassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce.ResultsThe effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.–vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p < 0.005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods.ConclusionOur method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions.

Elevated viral small RNA profiling in cassava cultivars suppress the occurrence of Cassava brown streak disease (CBSD)

Cassava brown streak disease (CBSD), caused by cassava brown streak virus (CBSV) and; Ugandan cassava brown streak virus (UCBSV) causes the most destructive cassava disease in Tanzania. Thus, breeders urgently need CBSD-resistant cultivars to combat the impact of this disease, safeguard cassava production, improve the yields and secure food supply for communities reliant on this staple crop. In this study, we used four cassava cultivars; Albert, KBH 2002/135, KBH 2006/026 and Kiroba to explore how viral small interfering RNA (vsiRNAs), an antiviral silencing mechanism, confers CBSD resistance. Cassava plants were inoculated by grafting method using the single infection of CBSV, UCBSV and mixed infection of these viruses (co-infection). Total RNA was extracted from leaves of each cultivar followed by deep sequencing. The result showed that high level of vsiRNAs was produced in inoculated plants, with the most prominent class being 21 and 22 nucleotides. Kiroba produced the highest level of vsiRNA in both CBSV and UCBSV, whereas KBH 2006/026 produced high level of vsiRNA only with UCBSV infections. In contrast to UCBSV, inoculation with CBSV stimulated severe symptoms but relatively low levels of vsiRNA. Co-infection treatment showed a more complex interaction between the host and virus, with variations in the severity and amount of vsiRNA produced. We conclude that resistance to CBSD varies depending on the type of cultivar and virus species, and the occurrence of CBSD is suppressed in plants with elevated vsiRNA. Therefore, a good understanding on the resistance status of parental materials for breeding is recommended to breeders as a basis for improving cassava production.

Genetic complexity of cassava brown streak disease: insights from qPCR-based viral titer analysis and genome-wide association studies.

Cassava, a vital global food source, faces a threat from Cassava Brown Streak Disease (CBSD). CBSD results from two viruses: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). These viruses frequently pose challenges to the traditional symptom-based 1-5 phenotyping method due to its limitations in terms of accuracy and objectivity. Quantitative polymerase chain reaction (qPCR) offers precise virus quantification, although high costs hinder its widespread adoption. In this research, we utilized qPCR to measure the viral titer/load of CBSV and UCBSV. The objectives were to evaluate titer variability within the Cycle 2 (C2) population in two different environments, establish connections between viral titers and CBSD severity scores from the 1-5 scoring method, perform Genome-Wide Association Studies (GWAS) to identify genomic regions associated with CBSV and UCBSV titers, and investigate the functional annotated genes. The results demonstrated a significantly higher prevalence of CBSV (50.2%) in clones compared to UCBSV (12.9%) with mixed infections in some cases. Genotypic effects, particularly concerning UCBSV, were significant, with genotype-by-environment effects primarily influencing CBSV titer. GWAS Studies identified genomic regions associated with CBSV and UCBSV titers. Twenty-one SNP markers on chromosomes 10, 13, 17, and 18 exhibited significant associations with CBSV titer, collectively explaining 43.14% of the phenotypic variation. Additionally, 25 SNP markers on chromosomes 1, 2, 4, 5, 8, 11, 12, 13, 16, and 18 were associated with UCBSV titer, and explained 70.71% of the phenotypic variation. No shared genomic regions were identified between CBSV and UCBSV viral titers. Gene ontology analysis also revealed diverse gene functions, especially in transport and catalytic activities. These findings enhance our understanding of virus prevalence, genetics, and molecular functions in cassava plants, offering valuable insights for targeted breeding strategies.

Going beyond consensus genome sequences: An innovative SNP-based methodology reconstructs different Ugandan cassava brown streak virus haplotypes at a nationwide scale inRwanda.

Cassava Brown Streak Disease (CBSD), which is caused by cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), represents one of the most devastating threats to cassava production in Africa, including in Rwanda where a dramatic epidemic in 2014 dropped cassava yield from 3.3 million to 900,000 tonnes (1). Studying viral genetic diversity at the genome level is essential in disease management, as it can provide valuable information on the origin and dynamics of epidemic events. To fill the current lack of genome-based diversity studies of UCBSV, we performed a nationwide survey of cassava ipomovirus genomic sequences in Rwanda by high-throughput sequencing (HTS) of pools of plants sampled from 130 cassava fields in thirteen cassava-producing districts, spanning seven agro-ecological zones with contrasting climatic conditions and different cassava cultivars. HTS allowed the assembly of a nearly complete consensus genome of UCBSV in twelve districts. The phylogenetic analysis revealed high homology between UCBSV genome sequences, with a maximum of 0.8 per cent divergence between genomes at the nucleotide level. An in-depth investigation based on Single Nucleotide Polymorphisms (SNPs) was conducted to explore the genome diversity beyond the consensus sequences. First, to ensure the validity of the result, a panel of SNPs was confirmed by independent reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Furthermore, the combination of fixation index (FST) calculation and Principal Component Analysis (PCA) based on SNP patterns identified three different UCBSV haplotypes geographically clustered. The haplotype 2 (H2) was restricted to the central regions, where the NAROCAS 1 cultivar is predominantly farmed. RT-PCR and Sanger sequencing of individual NAROCAS1 plants confirmed their association with H2. Haplotype 1 was widely spread, with a 100 per cent occurrence in the Eastern region, while Haplotype 3 was only found in the Western region. These haplotypes' associations with specific cultivars or regions would need further confirmation. Our results prove that a much more complex picture of genetic diversity can be deciphered beyond the consensus sequences, with practical implications on virus epidemiology, evolution, and disease management. Our methodology proposes a high-resolution analysis of genome diversity beyond the consensus between and within samples. It can be used at various scales, from individual plants to pooled samples of virus-infected plants. Our findings also showed how subtle genetic differences could be informative on the potential impact of agricultural practices, as the presence and frequency of a virus haplotype could be correlated with the dissemination and adoption of improved cultivars.

Detection of cassava brown streak ipomoviruses in aphids collected from cassava plants

Cassava is an important staple food in Africa and a major source of carbohydrates for 800 million people globally. However, cassava suffers severe yield losses caused by many factors including pests and diseases. A devastating disease of cassava is cassava brown streak disease (CBSD) caused by the cassava brown streak ipomoviruses (CBSIs) (family Potyviridae), Cassava brown streak virus (CBSV), and Ugandan cassava brown streak virus (UCBSV). Spread of CBSD is mainly through planting infected stem cuttings used for propagation. Transmission of CBSIs by the insect vector (Bemisia tabaci) has been reported. However, experimental transmission efficiencies of CBSIs are usually low. Recent research has showed the occurrence of a DAG motif associated with aphid transmission in other potyviruses, within the coat protein gene of CBSV. Consequently this study aimed to explore the possibility that besides whiteflies, aphids may transmit CBSIs. Cassava plants were assessed during a survey for occurrence of CBSD and aphids as potential alternative CBSIs vectors. We collected aphids from CBSD-symptomatic and symptomless cassava plants within farmers' fields in Uganda during April–July 2020. The aphids were analyzed for the presence of CBSIs by reverse transcriptase-polymerase chain reaction (RT-PCR) and to determine aphid species using mitochondrial cytochrome oxidase (mtCOI) barcoding. Unusual aphid infestation of cassava plants was observed at 35 locations in nine districts across Uganda and on 11 other plant species within or adjacent to cassava fields. This is the first report of aphids infesting cassava in Uganda. Molecular analysis of the aphid confirmed presence of three different aphid species in the surveyed cassava fields, namely, Aphis solanella, Aphis fabae mordvilkoi, and Rhopalosiphum sp. mtCOI nucleotide sequences for the aphids in which CBSIs were detected are deposited with Genbank under accession numbers OP223337-40. Both UCBSV and CBSV were detected by RT-PCR in aphids collected from cassava fields with CBSD-affected plants. The CBSIs were detected in 14 aphid samples collected from 19 CBSD-symptomatic cassava plants. These results suggest the ability of aphids to acquire CBSIs, but transmission experiments are required on their vector potential.

Survey of cassava brown streak disease and association of factors influencing its epidemics in smallholder cassava cropping systems of coastal Kenya

Cassava productivity is threatened by viral diseases which have become the main phytosanitary problems in cassava farmers. Cassava brown streak disease (CBSD) is a devastating viral disease caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) which are transmitted by whitefly vectors and mainly disseminated through the use of infected stem cuttings as planting materials. The aim of the current study was to: (1) assess farmers' knowledge, perceptions on spread, causes and current management practices of CBSD; (2) determine the factors associated with farmers' satisfaction with cassava planting material; and (3) determine the distribution, incidence, and severity of CBSD and association of factors influencing the disease epidemics in smallholder cassava cropping systems in coastal Kenya. Information was collected using semi-structured questionnaire administered to 250 smallholder farmers through face-to-face interviews coupled with field visits to assess the incidence, severity and distribution of CBSD. Symptomatic and asymptomatic cassava leaf samples were collected for reverse transcription-polymerase chain reaction (RT-PCR) analysis of the causal viruses of CBSD. The results revealed that majority of the farmers (96.6%) could recognize CBSD symptoms on the roots, and only 11.5% could recognize the foliar symptoms of the disease. The cause of the disease was unknown to the farmers, with no effective management methods available to them. Majority of farmers (82.5%) recycled own cassava cuttings from previous season's crop as planting material followed by exchanging/borrowing from neighbors (67.5%). The field incidence of CBSD was highest in Kilifi (27.9%) followed by Kwale (24.7%) and Taita Taveta (10.8%), with severities ranging from 2 to 3 in the three Counties. RT-PCR analysis indicated that 91% of the symptomatic samples tested positive for either of the two viruses occurring either singly or as dual infection. Approximately 3.2% of the asymptomatic samples tested positive for only CBSV. Findings from this study demonstrates the need for awareness creation of farmers on the causes, spread and management practices to control CBSD and the importance of strengthening certified cassava seed systems to reduce the impact of the disease. The study provides base-line information imperative for development of management strategies of CBSD.

Epidemiological Analysis of Cassava Mosaic and Brown Streak Diseases, and Bemisia tabaci in the Comoros Islands.

A comprehensive assessment of cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) was carried out in Comoros where cassava yield (5.7 t/ha) is significantly below the African average (8.6 t/ha) largely due to virus diseases. Observations from 66 sites across the Comoros Islands of Mwali, Ngazidja, and Ndzwani revealed that 83.3% of cassava fields had foliar symptoms of CBSD compared with 95.5% for CMD. Molecular diagnostics confirmed the presence of both cassava brown streak ipomoviruses (CBSIs) and cassava mosaic begomoviruses (CMBs). Although real-time RT-PCR only detected the presence of one CBSI species (Cassava brown streak virus, CBSV) the second species (Ugandan cassava brown streak virus, UCBSV) was identified using next-generation high-throughput sequencing. Both PCR and HTS detected the presence of East African cassava mosaic virus (EACMV). African cassava mosaic virus was not detected in any of the samples. Four whitefly species were identified from a sample of 131 specimens: Bemisia tabaci, B. afer, Aleurodicus dispersus, and Paraleyrodes bondari. Cassava B. tabaci comprised two mitotypes: SSA1-SG2 (89%) and SSA1-SG3 (11%). KASP SNP genotyping categorized 82% of cassava B. tabaci as haplogroup SSA-ESA. This knowledge will provide an important base for developing and deploying effective management strategies for cassava viruses and their vectors.

Screening of cultivars against cassava brown streak disease and molecular identification of the phytopathogenic infection-associated viruses

Cassava brown streak disease (CBSD) has been reported as a serious and significant constraint on the cassava crop in Democratic Republic of the Congo (DRC), especially in its eastern part, South Kivu, where cassava has exceedingly high socio-economic value in small-holder farming systems. The recent rapid spread and damage of CBSD are alarming. Compared to cassava mosaic disease (CMD) which causes yield losses of up to 70% in very susceptible cassava cultivars, CBSD destroys whole the field causing 100% crop loss, including immediate and direct losses of tuberous roots, planting material, and quality of leaves. The study gathered information on the circulation of viral species present in the region, and screening of cassava cultivars against CBSD. Here we evaluated the disease incidence (DI) and severity (DS), whitefly vector Bemisia tabaci parameters including number of nymphs and adults. Thereat, the work consisted of quantifying the pressure of the disease to find performing cassava cultivars, which may still be recommendable for farmers. Thirty-one improved and local Congolese cassava cultivars were selected. Disease assessment focused more specifically on morphological, epidemiologic studies, and advanced molecular diagnostic techniques for virus detection. The mean extreme values of disease incidence and severity ranged respectively from 0.0 to 59.44%, and 1 to 2.86, root infection up to 100%, whiteflies up to 2 adults, and 68 nymphs per plant. Accordingly, the viral species identified in circulation are Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (picornalike (+) ssRNA viruses, Ipomovirus, Potyviridae). Varieties like Cibongoyoka, Dorothea, Kamegere, Korengo, Liyayi, M’Bailo, Nabana, Rav, and Siri showed no symptoms of necrosis on the roots. Some cultivars are even less susceptible or not to the disease and may be recommended in this time of the lack of planting material for farmers.

Farmer-Cultivated Landraces and Improved Cassava Genotypes Exhibit Varied Response to Cassava Brown Streak Disease Under Natural Infections in Kenya

Cassava brown streak disease (CBSD) affects cassava growth and production with resultant foliar symptoms and storage root necrosis causing significant economic losses to farmers. The disease is caused by either Cassava brown streak virus (CBSV) or Uganda cassava brown streak virus (UCBSV), both vectored by whitefly, Bemisia tabaci. Cultivation of CBSD-free or resistant cassava genotypes offers the most long term sustainable CBSD management.

Cassava Brown Streak Viruses express second 6-kilodalton (6K2) protein with varied polarity and three dimensional (3D) structures: Basis for trait discrepancy between the virus species.

Cassava Brown Streak Virus (CBSV) and Ugandan Cassava Brown Streak Virus (UCBSV) are the two among six virus species speculated to cause the most catastrophic Brown Streak Disease of Cassava (CBSD) in Africa and Asia. Cassava Brown Streak Virus (CBSV) is hard to breed resistance for compared to Ugandan Cassava Brown Streak Virus (UCBSV) species. This is exemplified by incidences of CBSV species rather than UCBSV species in elite breeding line, KBH 2006/0026 at Bagamoyo, Tanzania. It is not yet understood as to why CBSV species could breakdown CBSD-resistance in the KBH 2006/0026 unlike the UCBSV species. This marks the first in silico study conducted to understand molecular basis for the trait discrepancy between CBSV and UCBSV species from structural biology view point. Following ab initio modelling and analysis of physical-chemical properties of second 6-kilodalton (6K2) protein encoded by CBSV and UCBSV species, using ROBETTA server and Protein Parameters tool, respectively we report that; three dimensional (3D) structures and polarity of the protein differs significantly between the two virus species. (95% and 5%) and (85% and 15%) strains of 20 CBSV and 20 UCBSV species respectively, expressed the protein in homo-trimeric and homo-tetrameric forms, correspondingly. 95% and 85% of studied strain population of the two virus species expressed hydrophilic and hydrophobic 6K2, respectively. Based on findings of the curent study, we hypothesize that; (i) The hydrophilic 6K2 expressed by the CBSV species, favour its faster systemic movement via vascular tissues of cassava host and hence result into higher tissue titres than the UCBSV species encoding hydrophobic form of the protein. t and (ii) The hydrophilic 6K2 expressed byCBSV species have additional interaction advantage with Nuclear Inclusion b protease domain (NIb) and Viral genome-linked protein (VPg), components of Virus Replication Complex (VRC) and hence contributing to faster replication of viral genome than the hydrophobic 6K2 expressed by the UCBSV species. Experimental studies are needed to resolve the 3D structures of the 6K2, VPg and NIb and comprehend complex molecular interactions between them. We suggest that, the 6K2 gene should be targeted for improvement of RNA interference (RNAi)-directed transgenesis of virus-resistant cassava as a more effective way to control the CBSD besides breeding.

Farmer and Field Survey in Cassava-Growing Districts of Rwanda Reveals Key Factors Associated With Cassava Brown Streak Disease Incidence and Cassava Productivity

Cassava (Manihot esculenta Crantz) is a vital crop in Rwanda where it ranks as the third most consumed staple. However, cassava productivity remains below its yield potential due to several constraints, including important viral diseases, such as cassava brown streak disease (CBSD). Because various factors can be addressed to mitigate the impact of viral diseases, it is essential to identify routes of virus contamination in the cassava agrosystems from the seed system to farmer's practices and knowledge. The present study aimed at (1) assessing the current cassava seed system and farmers' practices and their knowledge of the biotic constraints to cassava production, (2) determining the status of CBSD as well as critical factors associated with its spread through the seed system channels, and (3) determining factors that influence cassava productivity in Rwanda. A cross-sectional study was carried out from May to September 2019 in 13 districts of Rwanda. A total of 130 farmers and cassava fields were visited, and the incidence and severity of CBSD were evaluated. CBSD was detected in all cassava-producing districts. The highest field incidence of CBSD was recorded in the Nyanza district (62%; 95% CI = 56–67%) followed by the Bugesera district (60%; 95% CI = 54–65%), which recorded the highest severity score of 3.0 ± 0.6. RT-PCR revealed the presence of CBSD at the rate of 35.3%. Ugandan cassava brown streak virus was predominant (21.5%) although cassava brown streak virus was 4% and mixed infection was 10%. An informal cassava seed system was dominant among individual farmers, whereas most cooperatives used quality seeds. Cassava production was found to be significantly influenced by the use of fertilizer, size of the land, farming system, cassava viral disease, and type of cassava varieties grown (p &amp;lt; 0.001). Disease management measures were practiced by a half of participants only. Factors found to be significantly associated with CBSD infection (p &amp;lt; 0.05) were the source of cuttings, proximity to borders, age of cassava, and knowledge of CBSD transmission and management.

Expansion of the Cassava Brown Streak Disease Epidemic in Eastern Democratic Republic of Congo.

Cassava plays a key role in ensuring food security and generating income for smallholder farmers throughout Central Africa, particularly in the Democratic Republic of Congo (DRC). This status is threatened, however, by cassava brown streak disease (CBSD), which has expanded its incidence and range in eastern DRC. The study described here comprises the first extensive assessment of temporal change in the occurrence of CBSD and its causal viruses in DRC, based on surveys conducted during 2016 and 2018. Cassava fields were inspected in Ituri, Nord-Kivu, Sud-Kivu, Tanganyika, and Haut-Katanga provinces within eastern DRC to record foliar incidence and severity of CBSD. Leaf samples were collected for virus detection and species-level identification. New occurrences of CBSD, confirmed by virus diagnostic tests, were recorded in two provinces (Haut-Katanga and Sud-Kivu) and nine previously unaffected territories, covering an area of >62,000 km2, and at up to 900 km from locations of previously published reports of CBSD in DRC. Overall, average CBSD incidence within fields was 13.2% in 2016 and 16.1% in 2018. In the new spread zone of Haut-Katanga, incidence increased from 1.7 to 15.9%. CBSD is now present in provinces covering 321,000 km2, which is approximately 14% of the total area of DRC. This represents a major expansion of the CBSD epidemic, which was only recorded from one province (Nord-Kivu) in 2012. Both Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus were detected in Ituri, Nord-Kivu, and Sud-Kivu, but only CBSV was detected in Haut-Katanga. Overall, these results confirm the increasing threat that CBSD poses to cassava production in DRC and describe an important expansion in the African pandemic of CBSD.

Viral inosine triphosphatase: A mysterious enzyme with typical activity, but an atypical function.

Plant viruses typically have highly condensed genomes, yet the plant‐pathogenic viruses Cassava brown streak virus, Ugandan cassava brown streak virus, and Euphorbia ringspot virus are unusual in encoding an enzyme not yet found in any other virus, the “house‐cleaning” enzyme inosine triphosphatase. Inosine triphosphatases (ITPases) are highly conserved enzymes that occur in all kingdoms of life and perform a house‐cleaning function by hydrolysing the noncanonical nucleotide inosine triphosphate to inosine monophosphate. The ITPases encoded by cassava brown streak virus and Ugandan cassava brown streak virus have been characterized biochemically and are shown to have typical ITPase activity. However, their biological role in virus infection has yet to be elucidated. Here we review what is known of viral‐encoded ITPases and speculate on potential roles in infection with the aim of generating a greater understanding of cassava brown streak viruses, a group of the world's most devastating viruses.

Screening for Resistance in Farmer-Preferred Cassava Cultivars from Ghana to a Mixed Infection of CBSV and UCBSV.

Cassava brown streak disease (CBSD) caused by the Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is a threat to cassava production in Africa. The potential spread of CBSD into West Africa is a cause for concern, therefore screening for resistance in farmer-preferred genotypes is crucial for effective control and management. We multiplied a selection of eleven cassava cultivars grown by farmers in Ghana to test their response to a mixed infection of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) isolates using a stringent top-cleft graft inoculation method. Virus titers were quantified in the inoculated scions and cuttings propagated from the inoculated scions to assess virus accumulation and recovery. All cultivars were susceptible to the mixed infection although their response and symptom development varied. In the propagated infected scions, CBSV accumulated at higher titers in leaves of eight of the eleven cultivars. Visual scoring of storage roots from six-month-old virus-inoculated plants revealed the absence of CBSD-associated necrosis symptoms and detectable titers of CBSVs in the cultivar, IFAD. Although all eleven cultivars supported the replication of CBSV and UCBSV in their leaves, the absence of virus replication and CBSD-associated symptoms in the roots of some cultivars could be used as criteria to rapidly advance durable CBSD tolerance using breeding and genetic engineering approaches.

Genotype by environment cultivar evaluation for cassava brown streak disease resistance in Tanzania

Cassava brown steak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), is the most important biotic constraint to cassava production in East and Central Africa. Concerted efforts are required to prevent further spread into West Africa as well as to reduce losses in areas already affected. The study reported here was part of a five-country (Kenya, Malawi, Mozambique, Tanzania and Uganda) programme that aimed to identify superior cassava cultivars resistant to CBSD and to disseminate them widely in the region. Seventeen tissue-cultured and virus-tested cultivars were evaluated in Tanzania across nine sites with diverse CBSD inoculum conditions. Experiments were planted using an alpha-lattice design and assessments were made of surrounding inoculum pressure, CBSD foliar and root incidence and root yield at harvest. There were large differences in CBSD infection between sites, with greatest spread recorded from the north-western Lake (Victoria) zone. Differences were driven by Bemisia tabaci whitefly vector abundance and CBSD inoculum pressure. Both CBSV and UCBSV were almost equally represented in cassava fields surrounding experimental plots, although CBSV predominated in the north-west whilst UCBSV was more frequent in coastal and southern sites. However, the incidence of CBSV was much greater than that of UCBSV in initially virus-free experimental plots, suggesting that CBSV is more virulent. Cultivars could be categorised into three groups based on the degree of CBSD symptom expression in shoots and roots. The seven cultivars (F10_30R2, Eyope, Mkumba, Mkuranga1, Narocass1, Nase3 and Orera) in the most resistant category each had shoot and root incidences of less than 20%. Fresh root yield differed between sites and cultivars, but there was no genotype by environment interaction for this trait, probably attributable to the large fertility and soil moisture differences between sites. Susceptible cultivars and the local check performed well in the absence of CBSD pressure, highlighting the importance of exploiting quality and yield traits of local landraces in breeding programmes. Overall, our results emphasized the importance of applying a balanced strategy for CBSD management. This should use both improved and local germplasm resources to generate high yielding cultivars for specific end-user traits, and combine the deployment of improved cultivars with phytosanitary control measures including the use of healthy planting material and planting during periods of reduced CBSD infection.

Incidence, severity and distribution of Cassava brown streak disease in northeastern Democratic Republic of Congo.

Cassava fields were prospected from two provinces of the Democratic Republic of Congo (Ituri and Haut Uélé) to evaluate the ampleness of Cassava brown streak disease (CBSD) infection. CBSD pressure was determined by assessing the incidence, severity, whitefly abundance and distribution of the disease viruses in the surveyed provinces. A duplex RT-PCR was performed for the simultaneous detection of Ugandan Cassava brown streak virus (UCBSV) and Cassava brown streak virus (CBSV) on 56 cassava leaves sampled in the study area. Our results show a high field CBSD incidence contrasted to a low severity in both provinces. CBSD severity was similar in both provinces (mean disease severity 2). High densities of whitefly were recorded in Ituri province (10 adult whiteflies plant−1) than in Haut Uélé where density was 5 adults plant−1. However, no relation has been found between whitefly density and CBSD incidence and severity on cassava leaf, root and stems. Molecular analysis showed the incidence of single infections of UCBSV was greater than single infections of CBSV and mixed infections of UCBSV and CBSV. Disease incidence was greater in Ituri than in Haut Uélé; molecular incidence was lower than field incidence. Our results raise the need for appropriate CBSD control strategies in DRC.

Diversity and Phylogenetic Relationships of Full Genome Sequences of Cassava Brown Streak Viruses in Kenya

Cassava brown streak disease is caused by cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV). Many of the CBSV and UCBSV diversity studies utilize partial coat protein sequences due to the unavailability of representative full genome sequences. Hence, there is little information on the diversity of cassava brown streak viruses in the rest of the genomes of the two species that are present in the farmers’ fields. The aim of this study was to determine Kenyan full CBSV and UCBSV genomes, and their sequence diversity and phylogenetic relationships within various genome and genome segments. Twenty four CBSVs positive samples tested by RT PCR from major cassava producing regions in Kenya were sequenced using Illumina MiSeq. Quality assessment of the output reads was done using the CLC Genomics 5.5.1 software programs. Genome assembly was done by de novo and reference guided assembly. Nucleotide sequence similarity of CBSV and UCBSV was determined. Phylogenetic relationships between CBSV and UCBSV were determined by performing the neighbour-joining analysis using MEGA 6.0 software. Six CBSV and 9 UCBSV genomes were generated from this study. The coat protein of the CBSV sequences had nucleotide sequence similarity of 92-100% while P1 and P3 gene had 75-100% and 76-100%, respectively. The coat protein of the UCBSV sequences had nucleotide sequence similarity of 91-99%. P1 and P3 gene had 83-100% and 86-99%, respectively. The phylogenetic analysis of full genomes revealed two distinct clusters one for UCBSV and another cluster for CBSV. Individual gene segments phylogenetic tree resembled that of the whole genome by clustering the nucleotide sequences into two clusters, one belonging to UCBSV and the other CBSV. The study revealed an average genome nucleotide diversity of 21.5% and 15.8% in CBSV and UCBSV respectively. A vast genetic diversity observed in CBSV and UCBSV in this study portends a lot of challenges in developing molecular diagnostic techniques as well as control strategies against the viruses.

Utilization of infectious clones to visualize Cassava brown streak virus replication in planta and gain insights into symptom development

Cassava brown streak disease (CBSD) is a leading cause of cassava yield losses across eastern and central Africa and is having a severe impact on food security across the region. Despite its importance, relatively little is known about the mechanisms behind CBSD viral infections. We have recently reported the construction of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) infectious clones (IC), which can be used to gain insights into the functions of viral proteins and sequences associated with symptom development. In this study, we perform the first reporter gene tagging of a CBSV IC, with the insertion of green fluorescent protein (GFP) sequence at two different genome positions. Nicotiana benthamiana infections with the CBSV_GFP ICs revealed active CBSV replication in inoculated leaves at 2–5 days post inoculation (dpi) and systemic leaves at 10–14 dpi. We also constructed the chimera CBSV_UCP IC, consisting of the CBSV genome with a UCBSV coat protein (CP) sequence replacement. N. benthamiana infections with CBSV_UCP revealed that the CBSV CP may be associated with high levels of viral accumulation and necrosis development during early infection. These initial manipulations pave the way for U/CBSV ICs to be used to understand U/CBSV biology that will inform vital CBSD control strategies.

Cassava brown streak virus Ham1 protein hydrolyses mutagenic nucleotides and is a necrosis determinant.

SummaryCassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep‐sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild‐type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.

High-throughput sequencing of complete genomes of ipomoviruses associated with an epidemic of cassava brown streak disease in the Comoros Archipelago.

Using high-throughput sequencing of small interfering RNAs (siRNAs), virion-associated nucleic acid (VANA), and double stranded RNAs (dsRNAs), we have determined the complete genome sequences of Comorian isolates of two ipomoviruses, cassava brown streak virus (CBSV) and a divergent isolate of Ugandan cassava brown streak virus (UCBSV-KM) representing a new strain of this virus. While the large ORF of CBSV shares the highest nucleotide sequence identity (95.9%) with a Tanzanian isolate of CBSV, the large UCBSV-KM ORF shares the highest nucleotide sequence identity (77.5%) with a Malawian isolate of UCBSV. This low value is near the species demarcation threshold for the family Potyviridae (<76%). Phylogenetic analysis confirms that UCBSV-KM represents a new lineage that is genetically distinct from the currently described UCBSV strains.