The symptom and genetic diversity of cassava brown streak viruses infecting cassava in East Africa

The genetic and symptom diversity of six virus isolates causing cassava brown streak disease (CBSD) in the endemic (Kenya, Mozambique, and Tanzania) and the recently affected epidemic areas (Uganda) of eastern Africa was studied. Five cassava varieties; Albert, Colombian, Ebwanateraka, TMS60444 (all susceptible) and Kiroba (tolerant) were graft inoculated with each isolate. Based on a number of parameters including the severity of leaf and root symptoms, and the extent of virus transmission by grafting, the viruses were classified as either severe or relatively mild. These results were further confirmed by the mechanical inoculation of 13 herbaceous hosts in which the virulent isolates caused plant death in Nicotiana clevelandii and N. benthamiana whereas the milder isolates did not. Phylogenetic analysis of complete coat protein gene sequences of these isolates together with sequences obtained from 14 other field-collected samples from Kenya and Zanzibar, and reference sequences grouped them into two distinct clusters, representing the two species of cassava brown streak viruses. Put together, these results did not suggest the association of a hypervirulent form of the virus with the current CBSD epidemic in Uganda. Identification of the severe and milder isolates, however, has further implications for disease management and quarantine requirements

Attempts to identify Cassava Brown Streak Virus in western Democratic Republic of Congo

Open Access ArticleRoot necrosis similar to those of the cassava brown streak disease (CBSD) were observed on cassava in western provinces of the Democratic Republic of Congo (DR.Congo) in the early 2000’s. However molecular laboratory diagnosis were not able to detect any causative agent responsible for the attacks, hence, the disease related to these symptoms was named CBSD-like disease. In order to assess the distribution and the incidence of the CBSD-like disease, surveys were carried out in four western provinces, comprising, Kwango and Kwilu, Sud Ubangi, Kinshasa and Kongo Central. CBSD-like disease was observed in all surveyed provinces on the basis of root symptoms because foliar symptoms were different to those of the documented cases of CBSD in other parts of east Africa. CBSD-like disease incidence was high in Kongo Central and Sud Ubangi, exceeding an average of 50 %, but low in Kwango and Kwilu (32.8%) and in Kinshasa (19.1%). During the surveys, cassava leaf samples were collected for lab identification of the causal agent. PCR diagnosis was done on these samples using primers specific for the two known CBSVs. All samples tested negative with no amplification of DNA fragments of the correct size. Thus, further analysis on the causative organism is needed using Next Generation Sequencing (NGS) approaches. NGS approaches will help also to identify the causative organism in other Central Africa countries (Angola, Congo-Brazzaville and Gabon) where such cassava root necrosis have been reported or are suspected

Genome-wide analyses of cassava Pathogenesis-related (PR) gene families reveal core transcriptome responses to whitefly infestation, salicylic acid and jasmonic acid.

BACKGROUND:Whiteflies are a threat to cassava (Manihot esculenta), an important staple food in many tropical/subtropical regions. Understanding the molecular mechanisms regulating cassava's responses against this pest is crucial for developing control strategies. Pathogenesis-related (PR) protein families are an integral part of plant immunity. With the availability of whole genome sequences, the annotation and expression programs of the full complement of PR genes in an organism can now be achieved. An understanding of the responses of the entire complement of PR genes during biotic stress and to the defense hormones, salicylic acid (SA) and jasmonic acid (JA), is lacking. Here, we analyze the responses of cassava PR genes to whiteflies, SA, JA, and other biotic aggressors. RESULTS:The cassava genome possesses 14 of the 17 plant PR families, with a total of 447 PR genes. A cassava PR gene nomenclature is proposed. Phylogenetic relatedness of cassava PR proteins to each other and to homologs in poplar, rice and Arabidopsis identified cassava-specific PR gene family expansions. The temporal programs of PR gene expression in response to the whitefly (Aleurotrachelus socialis) in four whitefly-susceptible cassava genotypes showed that 167 of the 447 PR genes were regulated after whitefly infestation. While the timing of PR gene expression varied, over 37% of whitefly-regulated PR genes were downregulated in all four genotypes. Notably, whitefly-responsive PR genes were largely coordinately regulated by SA and JA. The analysis of cassava PR gene expression in response to five other biotic stresses revealed a strong positive correlation between whitefly and Xanthomonas axonopodis and Cassava Brown Streak Virus responses and negative correlations between whitefly and Cassava Mosaic Virus responses. Finally, certain associations between PR genes in cassava expansions and response to biotic stresses were observed among PR families. CONCLUSIONS:This study represents the first genome-wide characterization of PR genes in cassava. PR gene responses to six biotic stresses and to SA and JA are demonstrably different to other angiosperms. We propose that our approach could be applied in other species to fully understand PR gene regulation by pathogens, pests and the canonical defense hormones SA and JA

Unsuccessful Cassava Brown Streak Disease (CBSD) evaluation attempts in western Democratic Republic of Congo and implications with cassava root necrosis disease (CRND) etiology

Open Access ArticleCassava brown streak disease (CBSD) is the second most important virus disease after Cassava mosaic disease (CMD), infecting cassava (ManihotesculetaCrantz) in Africa. The disease is caused by two distinct viruses, Cassava brown streak virus [2, 3] and Ugandan Cassava brown streak virus (family, Potyviridae: genus, Ipomovirus). Transmission of CBSV from one plant to another is reported to occur through grafting CBSV-free with infected cuttings and subsequent dissemination by infected cuttings. The basic approach to control of CBSD is selecting planting material from symptomless mother plants. Graft inoculation is the most efficient and effective of the techniques for CBSD virus transmission and consequently cuttings are the most effective way of the disease spreading. In early 2000s, cassava root necrosis similar to those of CBSD were reported in western provinces of Democratic Republic of Congo (RDC) (Kinshasa and Kongo Central) and up to date PCR diagnoses did not detect any causal agent related to the observed symptoms and the disease which was still referred as ‘CBSD-like disease’. Due to lack of molecular data and the similarity of root symptoms with CBSD, the existence of a virus has always been suspected to be the cause of CBSD-like propagation. Thus, 2 field experiments were proposed in order to verify the existence of a systematic transmission of a possible CBSD related virus, knowing that CBSD viruses are transmitted efficiently by cuttings. The first trial focused on the field evaluation of CBSD – like infected and apparently uninfected planting materials, while the second trial involved the importation of tanzanian CBSD resistant genotypes for evaluation in INERA Mvuazi research center under CBSD-like infection conditions. Results of the first trial did not show a systemic transmission of any CBSD-like pathogen while CBSD-resistant parents involved in the second trial all succumbed to CBSD-like disease

TRANSIENT STUDIES ON RNA INTERFERENCE AND COAT PROTEIN-MEDIATED RESISTANCE TO CASSAVA BROWN STREAK DISEASE

Cassava (Manihot esculenta Crantz) is an important staple and cash crop in Africa, Latin America and Asia. In east and southern Africa, cassava brown streak disease (CBSD) caused by cassava brown streak virus (CBSV) is associated with significant losses in cassava production. Previously, the disease was prevalent only along coastal eastern and southern Africa, but it recently emerged in Uganda and is spreading rapidly in the country as well as in neighboring countries. Apart from a few cultivars that have shown tolerance to CBSV in Tanzania, no effective resistance to CBSV has been developed and deployed to date. The full genome sequence of CBSV is not yet known, but it is thought to be monopartite, linear, positive sense ssRNA, translated into a polyprotein that is further auto-cleaved into functional proteins with the capsid protein (CP) at the C-terminus. The present study aims to develop transient resistance to CBSV through CP-mediated protection and RNA interference (RNAi) strategies. The entire CBSV CP gene was used to express the CP and thereby trigger CP-mediated protection against CBSV. In addition, the full-length CP gene and its N- and C-terminal regions were used to generate three RNAi constructs, with RNAi-GFP as an internal control for transient studies in sap-inoculated GFP transgenic Nicotiana benthamiana. An efficient protocol for sap transmission of CBSV to N. benthamiana was also developed and used in transient protection studies of the constructs as proof-of-concept for control of CBSV using virus-derived resistance strategies in cassava. The constructs offered high levels of protection against CBSV and are highly recommended for use to transform cassava to generate CBSV resistant cassava plants for the farmers

The role of the whitefly, Bemisia tabaci (Gennadius), and farmer practices in the spread of cassava brown streak ipomoviruses

Cassava brown streak disease (CBSD) is arguably the most dangerous current threat to cassava, which is Africa's most important food security crop. CBSD is caused by two RNA viruses: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). The roles of the whitefly Bemisia tabaci (Gennadius) and farmer practices in the spread of CBSD were investigated in a set of field and laboratory experiments. The virus was acquired and transmitted by B. tabaci within a short time (5–10 min each for virus acquisition and inoculation), and was retained for up to 48 hr. Highest virus transmission (60%) was achieved using 20–25 suspected viruliferous whiteflies per plant that were given acquisition and inoculation periods of 24 and 48 hr, respectively. Experiments mimicking the agronomic practices of cassava leaf picking or the use of contaminated tools for making cassava stem cuttings did not show the transmission of CBSV or UCBSV. Screenhouse and field experiments in Tanzania showed that the spread of CBSD next to spreader rows was high, and that the rate of spread decreased with increasing distance from the source of inoculum. The disease spread in the field up to a maximum of 17 m in a cropping season. These results collectively confirm that CBSV and UCBSV are transmitted by B. tabaci semipersistently, but for only short distances in the field. This implies that spread over longer distances is due to movements of infected stem cuttings used for planting material. These findings have important implications for developing appropriate management strategies for CBSD

A Presumptive Developmental Role for a Sea Urchin Cyclin B Splice Variant

We show that a splice variant–derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation

Estimating epidemiological parameters from experiments in vector access to host plants, the method of matching gradients.

Estimation of pathogenic life-history values, for instance the duration a pathogen is retained in an insect vector (i.e., retention period) is of particular importance for understanding plant disease epidemiology. How can we extract values for these epidemiological parameters from conventional small-scale laboratory experiments in which transmission success is measured in relation to durations of vector access to host plants? We provide a solution to this problem by deriving formulae for the empirical curves that these experiments produce, called access period response curves (i.e., transmission success vs access period). We do this by writing simple equations for the fundamental life-cycle components of insect vectors in the laboratory. We then infer values of epidemiological parameters by matching the theoretical and empirical gradients of access period response curves. Using the example of Cassava brown streak virus (CBSV), which has emerged in sub-Saharan Africa and now threatens regional food security, we illustrate the method of matching gradients. We show how applying the method to published data produces a new understanding of CBSV through the inference of retention period, acquisition period and inoculation period parameters. We found that CBSV is retained for a far shorter duration in its insect vector (Bemisia tabaci whitefly) than had previously been assumed. Our results shed light on a number of critical factors that may be responsible for the transition of CBSV from sub- to super-threshold R0 in sub-Saharan Africa. The method is applicable to plant pathogens in general, to supply epidemiological parameter estimates that are crucial for practical management of epidemics and prediction of pandemic risk

Approaches to diagnosis and detection of cassava brown streak virus (Potiviridae: Ipomovirus) in fieldgrown cassava crop

Open Access JournalCassava brown streak disease (CBSD) has been a problem in the East African coastal cassava growing areas for more than 70 years. The disease is caused by successful infection with Cassava Brown Streak Virus (CBSV) (Family, Potyviridae: Genus, Ipomovirus). Diagnosis of CBSD has for long been primarily leaf symptoms-based. This is unreliable due to the irregular pattern and variability of the disease phenotype in roots and leaves. The suitable method to undertake reliable field diagnostic survey and derive acceptable analysis of the disease situation has never been standardized. Zigzag and diagonal approaches for disease assessment have been used successfully on other diseases infecting cassava such as Cassava mosaic disease but neither of them has ever been tested and proven suitable for CBSD assessment. In addition, the suitable sample for successful molecular detection of the causal virus has never been optimised. The number of samples to be collected from large plant stands which would be a true representation of the population has never been determined. The effect of sample bulking on possible detection or non detection of infection particularly when un-infected samples are combined with infected ones is not known. In this study, the comparative efficiencies of diagonal and zigzag approaches to CBSD field diagnosis were tested through surveys conducted in 20 randomly selected farmers’ fields in major cassava growing areas of the Coastal and Lake Zones in Tanzania. Using molecular diagnostic techniques, the plant parts which are suitable for Cassava brown streak virus (CBSV) detection were determined. Sample bulking was tested for rationalized laboratory detection of CBSV over large cassava stands. The study revealed that CBSD incidences and severities obtained using either diagonal or zigzag approach did not differ significantly. Suitable parts for CBSV detection were identified to be flowers, fruits, apical buds, young tender leaves, newly-opened leaves, youngest symptomatic leaves, the tender top-green portion of the stem and non-necrotic storage root tissues. CBSV was not detected in seeds. In bulked leaf samples, CBSV was detected from ratios of 1:1 up to 1:19 of CBSVinfected to CBSV-free tissues in cultivar Albert. It was concluded that either zigzag or diagonal can be used for CBSD field diagnosis. A choice of the suitable sample is of absolute necessity, and bulking of many samples for collective CBSV detection over a large crop stand is effective