Cassava Brown Streak Disease Research Articles

English

Cassava brown streak disease (CBSD) is the most devastating disease of cassava in southern, eastern and cntral Africa, and can cause up to 100% yield loss. Limited progress has been made in breeding for host plant resistance due to limited knowledge on the resistance variability to the disease. Reaction of promising cassava genotypes to CBSD in multi-environments are also unknown. Therefore, this study intended to: (1) Identify additional sources of resistance to CBSD; (2) Determine the stability of resistance to CBSD, and (3) mega-environments for screening resistance to CBSD. Field evaluation of 19 genotypes was conducted in RCBD with three replications at three agro-ecologies of Uganda for two cropping cycles. Additive Main Effects and Multiplicative Interaction (AMMI) and (GGE) biplot models were used to analyze genotype-environment interactions. Based on mean field reaction, the six best genotypes identified for resistance to CBSD were: TZ/06/140, TMS30572, TZ /06/130, N3/66/1, N3/58/1 with N3/104/3 and N3/66/1 being the most stable. While N3/66/1, N3/58/1 and N3/104/3, Mzungu and Kigoma Red were reported to be putative new sources of resistance to CBSD in Uganda. Genotypes (G), Environments (E), and GxE interactions were all significant, with no genotype exhibiting complete resistance. The significant result for GxE interaction to CBSD indicates the need for multi-environment screening and is suggestive of quantitative nature of CBSD resistance. Key words: Cassava brown streak disease; resistance, GxE interaction, discriminatory ability.

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.

English

Cassava (Manihot esculenta) is a vital food security crop and staple in Africa, yet cassava brown streak disease (CBSD) and cassava mosaic disease result in substantial yield losses. The aim of this study was to identify genes that have undergone positive selection during adaptive evolution, from CBSD resistant, tolerant and susceptible M. esculenta varieties and inter-specific hybrids, as well as a wild cassava species. Transcriptomes of 13 genotypes were sequenced and three genes with strong positive selection were detected (designated as EG2771, EG964 and EG5651).  Sequence variation for candidate genes in 18 different cassava genotypes was examined in relation to known response to CBSD and whitefly infection. Although, we cannot ascribe a selection pressure that was responsible for the observed positive selection with complete certainty at this stage, given the congruence of the pattern of particular alleles of our positively selected genes and the pattern of disease resistance of the cassava varieties we examined, it is likely that some protein variants coded by alleles of EG2771 and EG964 may be associated with CBSD and whitefly resistance responses. This warrants further investigation. Other alleles of our positively selected genes were likely influenced by domestication or some other unknown selective pressure.   Key words: Cassava, cassava brown streak disease (CBSD), resistant, tolerant, susceptible.

Influence of Host Plant Resistance and Disease Pressure on Spread of Cassava Brown Streak Disease in Uganda

Cassava brown streak disease (CBSD) is a major constraint to cassava production in Uganda. The disease is caused by two ipomovirus species: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak vi rus (UCBSV), both transmitted by the whitefly vector ( Bemisia tabaci ). Since the outbreak of the CBSD epidemic in Uganda in 2004, knowledge of its spread in the field is still limited. In this study, five cassava genotypes with varying levels of resistance to CBSD: TME 204 (susceptible), I92/0067, MH 97/2961, MH 96/0686 (moderately tolerant) and NASE 3 (tolerant) were used to evaluate the effect of genotype and prevailing disease pressure on CBSD spread in Uganda. The experiment was established in a random ized Complete Block Design (RCBD) in three sites of varying CBSD disease pressure: high (Wakiso), moderate (Kamuli) and low (Lira) in

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.

First report of cassava brown streak disease and associated Ugandan cassava brown streak virus in Mayotte Island

In November 2013, symptoms of yellowing on leaves and brown necrotic rot on tuberous roots (Fig. 1) were observed on different cassava landraces (Manihot esculenta) in Dembeni on Mayotte Island, a French Overseas Department in the southwest Indian Ocean. The symptoms, similar to the cassava brown streak disease described in East Africa, suggested the possible involvement of cassava brown streak viruses (Mbanzibwa et al., 2011). The expanding cassava brown streak disease epidemic in East Africa is caused by two ipomoviruses, Cassava brown streak virus (CBSV; Monger et al., 2010) and Ugandan cassava brown streak virus (UCBSV; Winter et al., 2010). To verify the identity of the causal agent, symptom-bearing leaf samples of 17 affected plants were collected in March and June 2014. Total RNA was extracted from these samples using the RNeasy Plant Mini Kit (Qiagen, France). For the detection of cassava brown streak viruses, a two-step RT-PCR using CBSDDF2 and CBSDDR primers was used (Mbanzibwa et al., 2011). These primers were designed for simultaneous virus-specific detection of CBSV and UCBSV (amplicons of 344 bp and 440 bp, respectively). RT-PCR products of the expected size for UCBSV were obtained for 11 samples. To confirm the diagnosis, the RT-PCR products were directly and bi-directionally sequenced and contigs assembled using DNABaser (Heracle BioSoft, Romania). Sequences of 376 bp from eight of the 11 samples were obtained which had 99-100% nucleotide identity (MEGA6; Tamura et al., 2013) (EMBL Accession Nos. LN611671 to LN611678; Table 1). The sequences showed the highest nucleotide identity (86%) with isolates of UCBSV from Uganda and Malawi (FN434109; FN433932) and 85% with an isolate of CBSV from Mozambique (FN434436). A phylogenetic reconstruction with publicly available complete genome sequences of cassava brown streak viruses confirmed that the Mayotte isolates are genetically more related to UCBSV isolates than to CBSV isolates from East Africa, and represent a separate lineage that appears to be almost intermediate between current isolates of CBSV and UCBSV (MEGA6; Fig. 2). This is the first report of cassava brown streak disease in Mayotte. Given that cassava is a staple food in the Comoros archipelago and Madagascar, this report is of great significance for managing the health of cassava in this region, and warrants urgent attention from regulatory institutions.

Genotype × environment interaction effects on early fresh storage root yield and related traits in cassava

Cassava (Manihot esculenta Crantz) is an important root crop worldwide. It exhibits substantial differential genotypic responses to varying environmental conditions, a phenomenon termed genotype×environment interaction (GEI). A significant GEI presents challenges in the selection of superior genotypes. The objective of this study was to examine the effect of genotype, environment and GEI on early fresh storage root yield (FSRY) and related traits in cassava. Accordingly, 12 cassava genotypes were evaluated in a randomised complete block design at three contrasting locations (Jinja, Nakasongola and Namulonge) in Uganda. Trials were harvested nine months after planting and the data collected were analysed using the additive main effects and multiplicative interaction (AMMI) model. The AMMI analysis of variance showed significant variation among genotypes for early FSRY and all other traits assessed. Locations were significantly different for all traits except for cassava brown streak disease root necrosis. The GEI effect was non-significant for early FSRY, but significant for other traits. For early FSRY, 48.5% of the treatment sum of squares was attributable to genotypes, 27.3% to environments, and 24.1% to GEI, indicating a predominance of genotypic variation for this trait. Predominance of genotypic variation was also observed for all the other traits. A majority of the genotypes (67%) had low interaction effects with locations for early FSRY, with Akena, CT2, CT4 and NASE14 being the most stable genotypes for the trait. Significant negative correlation was observed between cassava mosaic disease severity and early FSRY and storage root number, indicating significant negative effects of cassava mosaic disease on early FSRY and stability in cassava. The information generated will inform future selection initiatives for superior early-yielding cassava genotypes combining resistance to cassava mosaic and brown streak diseases in Uganda.

Biology and management of Bemisia whitefly vectors of cassava virus pandemics in Africa

Cassava mosaic disease and cassava brown streak disease are caused by viruses transmitted by Bemisia tabaci and affect approximately half of all cassava plants in Africa, resulting in annual production losses of more than $US 1 billion. A historical and current bias towards virus rather than vector control means that these diseases continue to spread, and high Bemisia populations threaten future virus spread even if the extant strains and species are controlled. Progress has been made in parts of Africa in replicating some of the successes of integrated Bemisia control programmes in the south-western United States. However, these management efforts, which utilise chemical insecticides that conserve the Bemisia natural enemy fauna, are only suitable for commercial agriculture, which presently excludes most cassava cultivation in Africa. Initiatives to strengthen the control of B. tabaci on cassava in Africa need to be aware of this limitation, and to focus primarily on control methods that are cheap, effective, sustainable and readily disseminated, such as host-plant resistance and biological control. A framework based on the application of force multipliers is proposed as a means of prioritising elements of future Bemisia control strategies for cassava in Africa.

Transcriptional response of virus-infected cassava and identification of putative sources of resistance for cassava brown streak disease.

Cassava (Manihot esculenta) is a major food staple in sub-Saharan Africa, which is severely affected by cassava brown streak disease (CBSD). The aim of this study was to identify resistance for CBSD as well as to understand the mechanism of putative resistance for providing effective control for the disease. Three cassava varieties; Kaleso, Kiroba and Albert were inoculated with cassava brown streak viruses by grafting and also using the natural insect vector the whitefly, Bemisia tabaci. Kaleso expressed mild or no disease symptoms and supported low concentrations of viruses, which is a characteristic of resistant plants. In comparison, Kiroba expressed severe leaf but milder root symptoms, while Albert was susceptible with severe symptoms both on leaves and roots. Real-time PCR was used to estimate virus concentrations in cassava varieties. Virus quantities were higher in Kiroba and Albert compared to Kaleso. The Illumina RNA-sequencing was used to further understand the genetic basis of resistance. More than 700 genes were uniquely overexpressed in Kaleso in response to virus infection compared to Albert. Surprisingly, none of them were similar to known resistant gene orthologs. Some of the overexpressed genes, however, belonged to the hormone signalling pathways and secondary metabolites, both of which are linked to plant resistance. These genes should be further characterised before confirming their role in resistance to CBSD.

Management of whitefly-transmitted viruses in open-field production systems.

Whiteflies are a key pest of crops in open-field production throughout the tropics and subtropics. This is due in large part to the long and diverse list of devastating plant viruses transmitted by these vectors. Open-field production provides many challenges to manage these viruses and in many cases adequate management has not been possible. Diseases caused by whitefly-transmitted viruses have become limiting factors in open-field production of a wide range of crops, i.e., bean golden mosaic disease in beans, tomato yellow leaf curl disease in tomato, cassava mosaic disease and cassava brown streak disease in cassava, and cotton leaf crumple disease in cotton. While host resistance has proven to be the most cost-effective management solution, few examples of host resistance have been developed to date. The main strategy to limit the incidence of virus-infected plants has been the application of insecticides to reduce vector populations aided to some extent by the use of selected cultural practices. However, due to concerns about the effect of insecticides on pollinators, consumer demand for reduced pesticide use, and the ability of the whitefly vectors to develop insecticide-resistance, there is a growing need to develop and deploy strategies that do not rely on insecticides. The reduction in pesticide use will greatly increase the need for genetic resistance to more viruses in more crop plants. Resistance combined with selected IPM strategies could become a viable means to increase yields in crops produced in open fields despite the presence of whitefly-transmitted viruses.

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.

Cassava Pests and Diseases’ Prevalence and Performance as Revealed by Adaptive Trial Sites in North Western Agro-Ecological Zone of Uganda

Cassava adaptive trial was planted in ten sub counties across West Nile Agro-ecological Zone (WNAEZ) with six cassava varieties to test their performance and reactions to major pests and diseases present in the Zone. Th six cassava varieties comprised of improved (TME 14 and 204, NASE 13 and 14 and Akena – TMS I92/00067) and a local (Abiria) were selected based on their availability and preference in the region. The experiment was planted in RCBD design with three replicates. The experiment was planted in ten locations with the plot size was 6x6 metres. Results showed Cassava green mite (CGM), Cassava mosaic disease (CMD) and cassava bacterial blight (CBB) as major pest and diseases across all sites. Cassava Anthracnose (CA) and Cassava mealy bug (CM) were not present in the study sites. Cassava brown streak disease (CBSD) foliar symptoms was observed in three locations of Dranya s/c, Gimara s/c and Nyaravuru s/c on the three varieties of TME 204, TMS-I92/00067 and TME 14, whereas CBSD root necrosis was seen across all sites on TME 204, TMS-I92/00067, NASE 13, TME 14 and the Local except on NASE 14. In terms of yields, results showed that it was highest in TMS-I92/00067 (53.0 t/ha), TME 204 (46.0 t/ha), NASE 14 (39.4 t/ha), TME 14 (34.6 t/ha), NASE 13 (33.4 t/ha) and the local (22.7 t/ha) in that order. Farmers’ ranking of the studied cassava varieties in order of preference was in the order of NASE 14, TME 204, TMS-I92/00067, TME 14, NASE 13 and the Local. In conclusion, absence of both foliar and root symptoms on NASE 14 across all sites indicated that this variety is still tolerant to CBSD and can still be multiplied for production in West Nile Agro-ecological Zone.

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.

Spatio-temporal patterns of genetic change amongst populations of cassava Bemisia tabaci whiteflies driving virus pandemics in East and Central Africa

The greatest current threat to cassava in sub-Saharan Africa, is the continued expansion of plant virus pandemics being driven by super-abundant populations of the whitefly vector, Bemisia tabaci. To track the association of putatively genetically distinct populations of B. tabaci with pandemics of cassava mosaic disease (CMD) and cassava brown streak disease (CBSD), a comprehensive region-wide analysis examined the phylogenetic relationships and population genetics of 642 B. tabaci adults sampled from cassava in six countries of East and Central Africa, between 1997 and 2010, using a mitochondrial DNA cytochrome oxidase I marker (780 bases). Eight phylogenetically distinct groups were identified, including one, designated herein as ‘East Africa 1’ (EA1), not previously described. The three most frequently occurring groups comprised >95% of all samples. Among these, the Sub-Saharan Africa 2 (SSA2) group diverged by c. 8% from two SSA1 sub-groups (SSA1-SG1 and SSA1-SG2), which themselves were 1.9% divergent. During the 14-year study period, the group associated with the CMD pandemic expansion shifted from SSA2 to SSA1-SG1. Population genetics analyses of SSA1, using Tajima's D, Fu's Fs and Rojas’ R2 statistics confirmed a temporal transition in SSA1 populations from neutrally evolving at the outset, to rapidly expanding from 2000 to 2003, then back to populations more at equilibrium after 2004. Based on available evidence, hybrid introgression appears to be the most parsimonious explanation for the switch from SSA2 to SSA1-SG1 in whitefly populations driving cassava virus pandemics in East and Central Africa.

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.

English

This study was conducted to: (1) determine the amount and structure of the genetic diversity of cassava in southern, eastern and central (SEC) Africa using simple sequence repeat (SSR) markers, (2) determine the frequency and distribution of alleles putatively associated with resistance to cassava brown streak disease (CBSD) derived from the variety ‘Namikonga’, and (3) examine the genetic relationships among some CBSD resistant genotypes. The major findings from this study were: (1) little genetic differentiation was confirmed among countries (7%) with the majority of variation distributed among individuals (93%), (2) the frequency of alleles putatively associated with resistance to CBSD was found to be generally low (0.2 to 0.5) but widely dispersed in the cassava germplasm of the SEC Africa region implying that if validated the markers will be useful for marker-assisted breeding on a broad scale, (3) several distinct putative sources of resistance to CBSD seem to be present in the cassava germplasm of SEC Africa offering the potential to pyramid genes for more effective and durable resistance and (4) it may be more informative to assess cassava diversity in the SEC Africa region based on pedigree (if known) or genetic relatedness, rather than geographical origin. Key words: Cassava brown streak disease (CBSD), simple sequence repeat (SSR), genetic diversity, pedigree information.

Genetic Relationships of Cassava Genotypes That are Susceptible or Tolerant to Cassava Brown Streak Disease in Uganda

A total of 99 cassava genotypes whose field reaction to cassava brown streak disease (CBSD) was known, were assayed with 30 simple sequence repeat (SSR) markers to establish their genetic parallels. Two categories of CBSD reaction were considered: CBSD-susceptible genotypes (characterized by > 60% root and foliar CBSD incidence), and CBSD-tolerant genotypes (characterized by < 15% root and foliar CBSD incidence). DNA was extracted from leaf samples using the miniprep method and genotyped using ABI 3730 DNA sequencer. The test genotypes at 0.02 similarity coefficient, CBSD-tolerant and CBSD-susceptible genotypes clustered into 5 main sub-clusters. When data were subjected to principle component analysis (PCA) irrespective of the CBSD reaction grade, the first three principal components accounted for 68% of the total genetic variation. Despite having different number of individuals, the observed heterozygosity (Ho) for CBSD-susceptible (Ho = 0.58) and CBSD-tolerant (Ho = 0.66) were comparable and provide scope for long-term CBSD breeding and/or gene tapping.

Isolation and characterization of resistant gene analogs in cassava, wild Manihot species, and castor bean (Ricinus communis)

Cassava, Manihot esculenta , is one of the major food crops in sub-Saharan Africa (SSA) providing the bulk of dietary calories to hundreds of millions of households. Two viral diseases, namely cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) pose a serious threat to cassava production. The emergence of new virus species and strains that overcome the existing resistant/tolerant cultivars entails identification and pyramiding of new sources of resistance using marker-aided selection. The isolation of resistance gene analogues (RGAs) using a homology-based approach can provide useful resources towards this goal. Degenerate primers based on the conserved motif of the nucleotide binding site (NBS) domain from resistance (R) genes were used to isolate RGAs from genomic DNA and cDNA in cassava, wild Manihot species, and castor bean ( Ricinus communis ). A total of 552 RGAs sequences were identified and deposited in GenBank. Conserved motifs such as P-loop, Kinase-2a and GLPL were present in the NBS domain. This study sheds light on the nature of NBS- leucine-rich repeat (LRR) R genes in cassava and closely related taxa in the family Euphorbiaceae. These candidate sequences mapped to the draft cassava genome with high sequence similarity to predicted NBS-LRR genes. These novel sequences may serve as a stepping stone for further characterization and experimental validation of predicted R genes in the draft cassava genome, ultimately leading to the development of functional gene-targeted markers that can be used in molecular resistance breeding aimed at combating CBSD and CMD. Key words: Cassava, resistance, Manihot , castor bean, resistance gene analog, nucleotide binding site.

Exploiting the combination of natural and genetically engineered resistance to cassava mosaic and cassava brown streak viruses impacting cassava production in Africa.

Cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) are currently two major viral diseases that severely reduce cassava production in large areas of Sub-Saharan Africa. Natural resistance has so far only been reported for CMD in cassava. CBSD is caused by two virus species, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). A sequence of the CBSV coat protein (CP) highly conserved between the two virus species was used to demonstrate that a CBSV-CP hairpin construct sufficed to generate immunity against both viral species in the cassava model cultivar (cv. 60444). Most of the transgenic lines showed high levels of resistance under increasing viral loads using a stringent top-grafting method of inoculation. No viral replication was observed in the resistant transgenic lines and they remained free of typical CBSD root symptoms 7 month post-infection. To generate transgenic cassava lines combining resistance to both CBSD and CMD the hairpin construct was transferred to a CMD-resistant farmer-preferred Nigerian landrace TME 7 (Oko-Iyawo). An adapted protocol allowed the efficient Agrobacterium-based transformation of TME 7 and the regeneration of transgenic lines with high levels of CBSV-CP hairpin-derived small RNAs. All transgenic TME 7 lines were immune to both CBSV and UCBSV infections. Further evaluation of the transgenic TME 7 lines revealed that CBSD resistance was maintained when plants were co-inoculated with East African cassava mosaic virus (EACMV), a geminivirus causing CMD. The innovative combination of natural and engineered virus resistance in farmer-preferred landraces will be particularly important to reducing the increasing impact of cassava viral diseases in Africa.

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.