Cassava Brown Streak Disease Symptoms Research Articles

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

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

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.

Smallholder Cassava Planting Material Movement and Grower Behavior in Zambia: Implications for the Management of Cassava Virus Diseases.

Cassava (Manihot esculenta) is an important food crop across sub-Saharan Africa, where production is severely inhibited by two viral diseases, cassava mosaic disease (CMD) and cassava brown streak disease (CBSD), both propagated by a whitefly vector and via human-mediated movement of infected cassava stems. There is limited information on growers' behavior related to movement of planting material, as well as growers' perception and awareness of cassava diseases, despite the importance of these factors for disease control. This study surveyed a total of 96 cassava subsistence growers and their fields across five provinces in Zambia between 2015 and 2017 to address these knowledge gaps. CMD symptoms were observed in 81.6% of the fields, with an average incidence of 52% across the infected fields. No CBSD symptoms were observed. Most growers used planting materials from their own (94%) or nearby (<10 km) fields of family and friends, although several large transactions over longer distances (10 to 350 km) occurred with friends (15 transactions), markets (1), middlemen (5), and nongovernmental organizations (6). Information related to cassava diseases and certified clean (disease-free) seed reached only 48% of growers. The most frequent sources of information related to cassava diseases included nearby friends, family, and neighbors, while extension workers were the most highly preferred source of information. These data provide a benchmark on which to plan management approaches to controlling CMD and CBSD, which should include clean propagation material, increasing growers' awareness of the diseases, and increasing information provided to farmers (specifically disease symptom recognition and disease management options).[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Developing dual‐resistant cassava to the two major viral diseases

AbstractCassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are two important biotic constraints affecting cassava (Manihot esculenta Crantz) production in sub‐Saharan Africa, and the deployment of cassava varieties dually resistant to both diseases is the most effective and realistic way of reducing losses. Crosses were carried out between a Tanzanian local cassava cultivar (Namikonga) and a South American cassava genotype (AR37‐80) to develop dual‐resistant progenies, and they were evaluated for two seasons at Naliendele in Southern Tanzania, which is a CMD and CBSD hot spot area. The CMD‐resistant progenies had low foliar severities (≤1.8), similar to the CMD‐resistant parent. The CBSD‐resistant progenies had minimal foliar (≤2.0) and root necrosis (≤1.2) severities, similar to the CBSD resistant parent, whereas CBSD‐tolerant progenies had severe foliar severities up to 3.3 but minimal root necrosis severities (≤1.2). Traits with minimal environmental influence also had high heritability (≥0.65) and high selection accuracy (≥0.70), and they included CMD foliar symptoms, CBSD foliar symptoms at 6 mo after planting, root necrosis, root necrosis incidence, root weight, root number per plant, and harvest index. Correlation analysis showed that the presence of disease reduces usable roots, root weight, root number per plant, and harvest index. Dual resistance can improve yield as observed in Namar 050 and Namar 371, which had high root weights of 27.5 and 28.2 t ha−1 with high genetic gains of 56.1 and 58.5%, respectively. Dual‐resistant progenies identified were Namar 050, Namar 100, Namar 130, Namar 200, Namar 334, Namar 371, and Namar 479, as they had minimal CMD and CBSD symptom severities (≤2.0) and could be used for breeding cassava varieties with superior characteristics.

Cassava whitefly species in eastern Nigeria and the threat of vector-borne pandemics from East and Central Africa.

Bemisia tabaci (sensu latu) is a group of >40 highly cryptic whitefly species that are of global agricultural importance, both as crop pests and plant-virus vectors. Two devastating cassava diseases in East and Central Africa are spread by abundant populations of one of these species termed Sub-Saharan Africa 1 (SSA1). There is a substantive risk that these whitefly-borne pandemics will continue to spread westwards and disrupt cassava production for millions of smallholder farmers in West Africa. We report here, therefore, the first comprehensive survey of cassava B. tabaci in eastern Nigeria, a West African region likely to be the first affected by the arrival of these whitefly-borne pandemics. We found one haplotype comprising 32 individuals with 100% identical mtCO1 sequence to the East African SSA1 populations (previously termed SSA1-SG1) and 19 mtCO1 haplotypes of Sub-Saharan Africa 3 (SSA3), the latter being the most prevalent and widely distributed B. tabaci species in eastern Nigeria. A more divergent SSA1 mtCO1 sequence (previously termed SSA1-SG5) was also identified in the region, as were mtCO1 sequences identifying the presence of the MED ASL B. tabaci species and Bemisia afer. Although B. tabaci SSA1 was found in eastern Nigeria, they were not present in the high abundances associated with the cassava mosaic (CMD) and cassava brown streak disease (CBSD) pandemics of East and Central Africa. Also, no severe CMD or any CBSD symptoms were found in the region.

Optimum Time for Harvesting Cassava Tubers to Reduce Losses Due to Cassava Brown Streak Disease in Northeastern DRC

The present study aimed to determine the appropriate time to harvest cassava tuberous root which minimize the losses due to cassava brown streak disease (CBSD) in the region of Yangambi, DRC. To achieve the aim of the study, 38 cassava cultivars were evaluated in Yangambi INERA&amp;rsquo;s Research Center for CBSD in the roots at harvest time between 9 and 13 months after planting (MAP). All the 38 cultivars tested showed CBSD root necrosis symptoms. Foliar symptoms occurred on 37.6% of the evaluated cultivars while CBSD root necrosis varied significantly among cultivars (7.0% to 82.5%) depending on susceptibility and the age of plant. This indicates the differential response of the cultivars to CBSD infection. &#x0D; &#x0D; Whitefly population density decreased with age of cassava, it was of 3 whiteflies per plant (9 MAP) to 1 (10 MAP). We noticed that in older plants, whitefly population decreased from 1 at 11 MAP to none at 13 MAP. Although, some cultivars did not show CBSD symptoms up to 12 MAP, they were not necessarily less attractive to whitefly. Negative relationship (r = -0.08 and r = -0.25) has been found between whitefly number and foliar symptom severity or between whitefly and tuber necrosis severity. Beyond 12 MAP, CBSD necrosis (severity score 4) was present in the tubers of 3 cultivars (EUR/2011/0148, Yafelamonene and Ybi/2011/258). Our study shows that in order to mitigate the losses due to CBSD necrosis, the optimum harvesting time for cassava tubers in Yangambi is 9 MAP.

Analyses of seven new whole genome sequences of cassava brown streak viruses in Mozambique reveals two distinct clades: evidence for new species.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Uganda cassava brown streak virus (UCBSV) is a major constraint to cassava production in Mozambique. Full genome sequences of CBSD‐associated virus isolates contribute to the understanding of genetic diversity and the development of new diagnostic primers that can be used for early detection of the viruses for sustainable disease management. This study determined seven new whole CBSV genomes from total RNA isolated from cassava leaves with CBSD symptoms collected from Nampula and Zambezia in Mozambique. Phylogenetic analyses of the new genomes with published CBSV and UCBSV sequences in GenBank grouped the CBSV isolates from Mozambique into two distinct clades together with CBSV isolates from Tanzania. Clade 1 and 2 isolates shared low nucleotide (79.1–80.4%) and amino acid (86.5–88.2%) sequence identity. Further, comparisons within the seven new CBSV isolates, and between them and the single published complete CBSV sequence (CBSV_MO_83_FN434436) from Mozambique, revealed nucleotide sequence identities of 79.3–100% and 79.3–98%, respectively, and amino acid identities of 86.7–100% and 86.7–98.8%. In addition, using RDP4, a recombination analysis comprising all CBSV and UCBSV genome sequences from GenBank detect 11 recombination events. Using several comprehensive evolutionary models and statistical programs, it was confirmed that CBSV and UCBSV are distinct virus species, with an additional probable new species (clade 2).

Response to Cassava Brown Streak Disease Infections in Local and Improved Cassava Genotypes under Field and Greenhouse Assays in Lower Eastern Kenya

Cassava brown streak disease (CBSD) is caused by two cassava brown streak viruses (CBSVs) transmitted by whiteflies (Bemisia tabaci). CBSD significantly inhibits cassava production in Kenya through losses of up to 100% in farmer-preferred but susceptible varieties. As a management strategy, the present study evaluated the effect of CBSD on two local varieties (Thika-5 &amp; Serere) and 15 improved genotypes in lower Eastern Kenya. Between October 2016 and June 2017, the genotypes were infected with CBSVs through whitefly transmission under field experiment at SEKU research farm (1.31ºS, 37.75ºE) and chip-bud grafting at KALRO-Katumani (1.35ºS, 37.14ºS) greenhouse conditions. RCBD and CRD experimental designs were respectively applied in field and greenhouse assays. CBSD symptoms were quantified through disease incidence (DIC) and severity (DSY) every 3 months for the field experiment and weekly for greenhouse assay. At harvest, storage root necrosis (SRN) was scored and non-necrotic roots weighed as marketable root yield (MRY). Molecular diagnostics was accomplished through duplex RT-PCR. Results revealed significantly (P≤0.01) higher foliar field DIC (81- 100%) and SRN (2.3 – 5.0) recorded in Thika-5 and Serere compared to all the improved genotypes that were foliarly asymptomatic (0% DIC and mean SRN of 1.0). Concomitantly and substantially lower (P≤0.01) MRY (1.99 – 2.16 t/ha) were bulked by Thika-5 and Serere compared to 10 improved genotypes that bulked 5.81 – 9.21 t/ha MRY. Upon chip-bud graft infection, Thika-5 and Serere showed higher DIC of 81 – 90% compared to four improved genotypes with 20 - 35% DIC. Correlations between MRY, DIC and SRN were inverse and significant (P≤0.01). RT-PCR detected pre-dominantly CBSV. In conclusion, the natural whitefly-based transmission of CBSVs was ineffective compared to chip-bud grafting. The inverse correlations between CBSD symptoms and yield corroborated the deleterious impact of CBSD on cassava production. The ten improved, high yielding and asymptomatic genotypes identified in the current study could potentially be used to confer resistance against CBSD into farmer-preferred but often sensitive varieties.

Simultaneous CRISPR/Cas9-mediated editing of cassava eIF4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence.

SummaryCassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive‐sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome‐linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E‐1, eIF(iso)4E‐2, novel cap‐binding protein‐1 (nCBP‐1), and nCBP‐2. Protein–protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9‐mediated genome editing was employed to generate ncbp‐1, ncbp‐2, and ncbp‐1/ncbp‐2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp‐1/ncbp‐2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild‐type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.

Plot based heritability estimates and categorization of cassava genotype response to cassava brown streak disease

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is a threat to food security in sub-Saharan Africa, where the disease persistently reduces overall root quality and quantity resulting in up to 100% yield losses. Complexities in CBSD symptom expression and the damage caused on leaves, stems and roots throughout the 12 months of cassava growth require that appropriate ways of categorizing genotype response and optimal stages of evaluation be identified. This study aimed at: 1) determining plot based heritability of CBSD based on symptom expression and 2) categorizing genotype resistance to CBSD based on symptom expression. Herein, 41 genotypes were evaluated for two years at Namulonge with an additional evaluation conducted across three locations. Evaluations were done at three, six, nine and twelve months after planting. Genotype responses to CBSD varied significantly. High broad sense heritability estimates of up to 0.81 (incidence) and 0.71 (severity) were obtained.Average disease severity scores had higher broad sense heritability estimates (0.53 and 0.65) than maximum disease severity scores (0.33 and 0.61) for root and foliar severities respectively. These findings are important in choosing an appropriate evaluation method for CBSD. Genotypes displayed differing CBSD responses in type, locality and severity of symptoms. This suggested that genotypes had differences in mechanisms of resistance that can be exploited in CBSD resistance breeding.

Cassava brown streak disease in Rwanda, the associated viruses and disease phenotypes.

Cassava brown streak disease (CBSD) was first observed on cassava (Manihot esculenta) in Rwanda in 2009. In 2014 eight major cassava‐growing districts in the country were surveyed to determine the distribution and variability of symptom phenotypes associated with CBSD, and the genetic diversity of cassava brown streak viruses. Distribution of the CBSD symptom phenotypes and their combinations varied greatly between districts, cultivars and their associated viruses. The symptoms on leaf alone recorded the highest (32.2%) incidence, followed by roots (25.7%), leaf + stem (20.3%), leaf + root (10.4%), leaf + stem + root (5.2%), stem + root (3.7%), and stem (2.5%) symptoms. Analysis by RT‐PCR showed that single infections of Ugandan cassava brown streak virus (UCBSV) were most common (74.2% of total infections) and associated with all the seven phenotypes studied. Single infections of Cassava brown streak virus (CBSV) were predominant (15.3% of total infections) in CBSD‐affected plants showing symptoms on stems alone. Mixed infections (CBSV + UCBSV) comprised 10.5% of total infections and predominated in the combinations of leaf + stem + root phenotypes. Phylogenetic analysis and the estimates of evolutionary divergence, using partial sequences (210 nt) of the coat protein gene, revealed that in Rwanda there is one type of CBSV and an indication of diverse UCBSV. This study is the first to report the occurrence and distribution of both CBSV and UCBSV based on molecular techniques in Rwanda.

Genotype by environment interactions in identifying cassava (Manihot esculenta Crantz) resistant to cassava brown streak disease

Cassava landraces were evaluated for resistance to cassava brown streak disease (CBSD) for two cropping seasons at a disease hotspot area in Naliendele, Tanzania. Based on reactions to CBSD, several landraces including Chimaje, Mfaransa and Supa B were considered to be resistant to the disease while Kikwada, Mbuyu, and Nyoka were tolerant. ANOVA revealed that the largest sum of squares (SS) (41.9–86.7%) was attributed to the genotype of the cassava landraces, while a smaller proportion of SS (8.1–38.2%) was due to genotype by environment interactions for all traits tested, which included disease symptoms, root weight, number of roots per plant and dry matter content. Environment accounted for the smallest effect (0.01–26.3%), however, the mean squares was nonetheless significant for a few genotypes, which indicated that their disease expression was indeed influenced by the environment. Increased CBSD severity was associated with low temperatures and rainfall. Increased rainfall towards harvesting led to higher root weight but lower dry matter content in the first cropping season. Correlation analysis showed that the presence of CBSD symptoms reduces the amount of usable roots, total root weight, and root dry matter content. Many resistant/tolerant landraces also had high root weight and dry matter content, and they can be used by farmers to reduce CBSD losses. The landraces described here form novel sources of CBSD resistance that can be used for breeding disease-resistant cassava varieties with superior agronomic characteristics.

Analysis of Interactions Between Cassava Brown Streak Disease Symptom Types Facilitates the Determination of Varietal Responses and Yield Losses.

Cassava brown streak disease (CBSD), caused by cassava brown streak viruses, is recognized as one of the most important plant disease threats to African food security. This study describes the incidence and severity of the different symptom types caused by CBSD, derived from extensive surveys in the country most severely affected by the disease: Tanzania. Total plant incidence and mean root severity of CBSD, recorded from 341 farmers' fields, were both greater in the Coast Zone (49.5% and 3.05), than in the Lake Zone (32.7% and 2.57). Overall, the differing incidences recorded declined in the following order: total plant incidence (39.1%), plant shoot incidence (33.4%), plant root incidence (19.3%), root incidence (10.5%), and unusable root incidence (5.4%). The much lower-than-anticipated loss due to the root necrosis that is characteristic of CBSD was offset by large reductions of root number in plants expressing foliar symptoms of CBSD (15.7% in the Coast Zone and 5.5% in the Lake Zone). These data suggest that the effects of CBSD on the growth of affected plants are greater than those due to root spoilage. Based on these two factors, annual losses due to CBSD in the parts of Tanzania surveyed were estimated at >860,000 t, equivalent to more than U.S.$51 million. A novel approach to using farm-derived data on the responses of the most frequently cultivated varieties to CBSD infection allowed the grouping of the varieties into four categories, based on their relative resistance or tolerance to infection. This tool should be of value to breeders in identifying and selecting for sources of resistance or tolerance in both local and exotic germplasm, and should ultimately contribute to enhancing the management of one of Africa's most damaging crop diseases.

Distribution and accumulation of cassava brown streak viruses within infected cassava (Manihot esculenta) plants

Cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), ranks among the top seven biological threats to global food security. The disease poses a significant threat to cassava production in East and Central Africa (ECA). In Uganda, overall CBSD incidence increased by c. 20% since it re‐emerged in 2004, and the disease persistently reduces cassava yields and storage root qualities. The spread of CBSD has been studied spatially in fields in different agroecologies. However, within‐host distribution and accumulation of CBSV and UCBSV in naturally infected cassava plants is unknown. Therefore, within‐host CBSV and UCBSV distribution was studied to correlate CBSD symptoms with virus titre in organs of infected cassava. Leaf, stem and storage root samples, with and without symptoms, were collected from 10 genotypes of field‐grown cassava. Presence of CBSV and UCBSV was detected by RT‐PCR and virus levels determined by qRT‐PCR. CBSV was present in 100% of CBSD samples with symptoms, with 45·3% positive for presence of both CBSV and UCBSV. Tolerant cassava genotypes were infected with CBSV alone and accumulated higher titre in roots than in aerial organs. Susceptible genotypes were co‐infected with CBSV and UCBSV and exhibited variation in virus titre in each organ. Across genotypes, virus titre was lowest in the youngest leaves and highest in mature non‐senescing leaves. This information provides insight into the relationship between CBSV, UCBSV and their cassava host, and is valuable for CBSD resistance breeding, epidemiology studies and CBSD control.

Field evaluation of selected cassava genotypes for cassava brown streak disease based on symptom expression and virus load.

BackgroundProduction of cassava (Manihot esculenta Crantz), a food security crop in sub-Saharan Africa, is threatened by the spread of cassava brown streak disease (CBSD) which manifests in part as a corky necrosis in the storage root. It is caused by either of two virus species, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), resulting in up to 100% yield loss in susceptible varieties.MethodsThis study characterized the response of 11 cassava varieties according to CBSD symptom expression and relative CBSV and UCBSV load in a field trial in Uganda. Relative viral load was measured using quantitative RT-PCR using COX as an internal housekeeping gene.ResultsA complex situation was revealed with indications of different resistance mechanisms that restrict virus accumulation and symptom expression. Four response categories were defined. Symptom expression was not always positively correlated with virus load. Substantially different levels of the virus species were found in many genotypes suggesting either resistance to one virus species or the other, or some form of interaction, antagonism or competition between virus species.ConclusionsA substantial amount of research still needs to be undertaken to fully understand the mechanism and genetic bases of resistance. This information will be useful in informing breeding strategies and restricting virus spread.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-014-0216-x) contains supplementary material, which is available to authorized users.

English

The cassava brown streak disease (CBSD) has spread rapidly in Northern Uganda since its emergence in Uganda in 2005. Field surveys conducted in 2010 and 2011 by Ngetta Zonal Agricultural Research and Development Institute (Ngetta ZARDI) revealed high CBSD prevalence in the zone. CBSD epidemic has severely affected livelihood of smallholder farmers in the region. Lack of knowledge on disease recognition and management contributed significantly to rapid spread of CBSD in the zone. Addressing this aspect to increase and improve the knowledge base of cassava farmers was an important component in integrated disease management. Ngetta ZARDI through the Agricultural Technology Agribusiness and Advisory Services (ATAAS) project developed a multi-displinary effort to reduce the spread and impact of CBSD, partly through promotion of adoption of tolerant cassava varieties, and educating and training of smallholder farmers and other stakeholders. Participants were carefully selected from farmer groups, extension and political leadership. Training involved lectures and field visits for practical demonstration of CBSD symptoms. The training increased the number of people with knowledge on CBSD management up to farmer level. Overall, the training program significantly strengthened the human resource base employed against CBSD in the zone. Key words: Cassava brown streak disease, Northern Uganda.

Efficient transmission of Cassava brown streak disease viral pathogens by chip bud grafting

BackgroundTechniques to study plant viral diseases under controlled growth conditions are required to fully understand their biology and investigate host resistance. Cassava brown streak disease (CBSD) presents a major threat to cassava production in East Africa. No infectious clones of the causal viruses, Cassava brown streak virus (CBSV) or Ugandan cassava brown streak virus (UCBSV) are available, and mechanical transmission to cassava is not effective. An improved method for transmission of the viruses, both singly and as co-infections has been developed using bud grafts.FindingsAxillary buds from CBSD symptomatic plants infected with virulent isolates of CBSV and UCBSV were excised and grafted onto 6–8 week old greenhouse-grown, disease-free cassava plants of cultivars Ebwanateraka, TME204 and 60444. Plants were assessed visually for development of CBSD symptoms and by RT-PCR for presence of the viruses in leaf and storage root tissues. Across replicated experiments, 70-100% of plants inoculated with CBSV developed CBSD leaf and stem symptoms 2–6 weeks after bud grafting. Infected plants showed typical, severe necrotic lesions in storage roots at harvest 12–14 weeks after graft inoculation. Sequential grafting of buds from plants infected with UCBSV followed 10–14 days later by buds carrying CBSV, onto the same test plant, resulted in 100% of the rootstocks becoming co-infected with both pathogens. This dual transmission rate was greater than that achieved by simultaneous grafting with UCBSV and CBSV (67%), or when grafting first with CBSV followed by UCBSV (17%).ConclusionsThe bud grafting method described presents an improved tool for screening cassava germplasm for resistance to CBSD causal viruses, and for studying pathogenicity of this important disease. Bud grafting provides new opportunities compared to previously reported top and side grafting systems. Test plants can be inoculated as young, uniform plants of a size easily handled in a small greenhouse or large growth chamber and can be inoculated in a controlled manner with CBSV and UCBSV, either singly or together. Disease symptoms develop rapidly, allowing better studies of interactions between these viral pathogens, their movement within shoot and root systems, and how they induce their destructive disease symptoms.

RNAi-derived field resistance to Cassava brown streak disease persists across the vegetative cropping cycle

A confined field trial was established to determine durability of RNAi-mediated resistance to Cassava brown streak disease (CBSD). Stem cuttings were obtained from field-grown cassava plants of cv 60444 transgenic for construct p718, consisting of an 894 bp inverted repeat sequence from the Ugandan Cassava brown streak virus (UCBSV) coat protein. Plants were established from three transgenic lines previously shown to provide complete resistance to UCBSV and differing levels of protection to the non-homologous virus species Cassava brown streak virus (CBSV), and grown for 11 months. CBSD symptoms were observed on shoots and storage roots of all non-transgenic cv 60444 control plants and transgenic lines p718–002 and p718–005, but not on p718–001. RT-PCR diagnostic showed tissues of plant lines p718–002 and p718–005 to be infected with CBSV, but free of UCBSV. All leaves and roots of p718–001 plants were to carry no detectable levels of either pathogen. Plants of cv 60444 in this field trial showed severe cassava mosaic disease symptoms, indicating that presence of replicating geminiviruses did not cause significant suppression of RNAi-mediated resistance to CBSD. Resistance to CBSD across a vegetative cropping cycle confirms earlier field data, and provides an important step in proof of concept for application of RNAi technology to control of CBSD under conditions encountered in farmers’ fields.

Transgenic RNA interference (RNAi)‐derived field resistance to cassava brown streak disease

Cassava brown streak disease (CBSD), caused by the Ipomoviruses Cassava brown streak virus (CBSV) and Ugandan Cassava brown streak virus (UCBSV), is considered to be an imminent threat to food security in tropical Africa. Cassava plants were transgenically modified to generate small interfering RNAs (siRNAs) from truncated full-length (894-bp) and N-terminal (402-bp) portions of the UCBSV coat protein (ΔCP) sequence. Seven siRNA-producing lines from each gene construct were tested under confined field trials at Namulonge, Uganda. All nontransgenic control plants (n = 60) developed CBSD symptoms on aerial tissues by 6 months after planting, whereas plants transgenic for the full-length ΔCP sequence showed a 3-month delay in disease development, with 98% of clonal replicates within line 718-001 remaining symptom free over the 11-month trial. Reverse transcriptase-polymerase chain reaction (RT-PCR) diagnostics indicated the presence of UCBSV within the leaves of 57% of the nontransgenic controls, but in only two of 413 plants tested (0.5%) across the 14 transgenic lines. All transgenic plants showing CBSD were PCR positive for the presence of CBSV, except for line 781-001, in which 93% of plants were confirmed to be free of both pathogens. At harvest, 90% of storage roots from nontransgenic plants were severely affected by CBSD-induced necrosis. However, transgenic lines 718-005 and 718-001 showed significant suppression of disease, with 95% of roots from the latter line remaining free from necrosis and RT-PCR negative for the presence of both viral pathogens. Cross-protection against CBSV by siRNAs generated from the full-length UCBSV ΔCP confirms a previous report in tobacco. The information presented provides proof of principle for the control of CBSD by RNA interference-mediated technology, and progress towards the potential control of this damaging disease.

Re-emergence of Cassava Brown Streak Disease in Uganda.

During November 2004, veinal chlorosis on mature cassava leaves, typical of cassava brown streak disease (CBSD), was observed at Mukono in central Uganda. Five out of 11 cultivars at the site showed CBSD symptoms (incidence range 4 to 64%). In a survey of farmers' fields, CBSD was observed in Wakiso and Mukono districts. Incidence of cassava mosaic disease was also recorded and averaged 60% for landraces (range 16.7 to 100%) and 20% for resistant varieties (range 0 to 65%). Leaf samples of plants with CBSD symptoms produced an amplicon of 222 bp using reverse transcription-polymerase chain reaction with primers that amplify a fragment of the coat protein (CP) gene of Cassava brown streak virus. Sequence comparisons based on the amplified CP gene fragment indicated that the isolates have 77 to 82.9% nucleotide and 43.9 to 56.8% amino acid identity with those from Mozambique and Tanzania. There was 95.9 to 99.5% nucleotide and 85.1 to 90.5% amino acid identity among the Ugandan isolates. These results confirm the re-emergence of CBSD in Uganda after it was first observed in the 1930s in cassava introduced from Tanzania and controlled by eradication. Prior to this report, CBSD was known to be restricted to the coastal lowlands of East Africa.