Attempts to Identify Cassava Brown Streak Virus in Western Democratic Republic of Congo

Root 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.


Introduction
Cassava (Manihot esculenta Crantz) is one of the food crops that is extensively cultivated in the Sub-Saharan Africa.Its total production is estimated at more than 121 millions of tons, which is more than any other crop in Africa (FAO, 2006) and 54 % of the world production (FAO, 2003).
Cassava is very important for small holder farmers because of its role in food security and as source of incomes (Fauquet & Fargette, 1990;Harrison et al., 1997).However, cassava yields still remains low at 8-9 tons/ha which is about 70 % of what is obtained in South America and 61% in Asia (Legg & Thresh, 2003).The total production of cassava in DR, Congo was recorded at 15,020,000 tons (FAOSTAT, 2009).
Cassava production in Africa is mostly constrained by pests and diseases (Hahn & Keyser, 1985).Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) (Geminiviridae; Begomovirus) is undoubtedly the most important constraint to the production of cassava in Africa at the outset of the 21st century (Legg & Fauquet, 2004).
The most visible symptom of the disease is the expression of the characteristic leaf mosaic, and young plants are more severely affected than old ones.Symptoms range from barely perceptible mosaic to stunting of the plant and extreme reduction of the leaf blades (Fauquet & Fargette, 1990).
In the 2000s, DR, Congo experienced a severe outbreak of the CMD by the Ugandan variant (EACMV-UG) which was detected in most of cassava growing areas in the country resulting in significant crop losses (Lema et al., 2004;Tata-Hangy et al., 2004).
Cassava brown streak disease (CBSD) is another viral disease that constrains cassava production in most parts of East African region.Hillocks et al., 2001 estimated loss in the fresh roots yield due to CBSD at more than 70%.It was recognized for long as being endemic in the inshore of cassava areas in the coastal zones (Storey, 1936;Rwegasira et al., 2011) and limited to low and mid altitudes below 1000 meters (Nichols, 1950).However, from 2004, outbreaks were reported at altitudes greater than 1000 meters in the Great lakes region of east and central Africa (Alicai et al., 2007).Currently, CBSD is occurring in various eastern and southern African countries including Kenya, Malawi, Mozambique, Tanzania and Uganda (Alicai et al., 2007).More recently, CBSD has been reported in eastern DR, Congo and confirmed at molecular level (Mulimbi et al., 2012).
Leaf symptoms include blotchy yellow chlorosis or feathery necrosis, often associated with minor veins, which can appear within the first few months after planting of infected cuttings and persist in mature leaves.Brown, round or elongate streak-like lesions can occur on the young green portion of infected stems, but the main economic loss is caused by dry, brown necrotic lesions in the storage tissues of the tuberous roots of infected susceptible plants (Storey, 1936;Legg et al., 2011).Root constrictions are also sometimes observed as well as brown/black lesions on green fruits, and necrotic lesions in leaf scars.In severe infections these lesions develop to kill the dormant axilliary buds leading to a general shrinkage of the node and death of the intermodal tissue, so that the branch dies from the tip to cause 'dieback' (Hillocks & Jennings, 2003).Secondary losses occur as a consequence of early harvesting, which farmers use as a strategy to avoid root necrosis (Hillocks, 2003).
CBSD causes economic damages on tuberous roots in form of yellow/brown necrosis bearing tissues which became unfit for human consumption (Hillocks & Jennings, 2003).
CBSD is known to be caused by a RNA (ssRNA) virus of the family Potyviridae; genus Ipomovirus (Monger et al., 2001a).Two genetically distinct strains of CBSVs were recognized in East Africa (Mbazibwa et al., 2009), which are shown to be two distinct species, Cassava brown streak virus (classic) and Ugandan cassava brown streak virus (Monger et al., 2010).
In early 2000s, cassava root necrosis (Figure 1) similar to those of CBSD were reported in western provinces of DR.Congo (Kinshasa and Kongo Central) by Mahungu et al. (2003) and up to date PCR diagnoses did not detect any causal agent related to the observed symptoms and the disease is still referred to as 'CBSD-like disease'.jas.ccsenet.

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Using molecular analysis, Mulimbi et al. (2012) confirmed CBSV (more specifically the UCBSV) to be present in eastern DR, Congo.All our results CBSV PCR assays are conform to all previous negative results from earlier investigators in western DR, Congo.

Discussion
Results on the distribution and incidence of the CBSD-like disease in the four surveyed provinces in western DR, Congo suggest that more attention needs to be given in the immediate future to the potential risk of this new type of CBSD or new disease in the country.Urgent interventions are needed, based first on the identification of the causal agent, and further, on the mitigation of the disease that will consists in the development of resistant varieties as a major component of an integrated management approach.
Deep sequencing of freshly collected infected cassava leaves using next-generation sequencing of polyA + RNA and small RNAs should be recommended to elucidate the causes of CBSD-like symptoms in western DR, Congo.This approach has also the potential to discover novel viruses.
Since these viruses have only recently been recognized, descriptions of further CBSD causing viruses can be expected (Ndunguru et al., 2015).Robertson et al. (1991) and Omunyin et al. (1996) stated that based on the nucleotide sequence information of several different viruses, specific primers are designed and used in PCR to detect and differentiate viruses at the family, genus, or strain level.Also, simultaneous detection of unrelated viruses in a sample by using a mixture of virus-specific primer is possible ("Multiplex" PCR) (Bariana et al., 1994;Abarshi et al., 2012).
It is known also that the polyphagous nature of B. tabaci may contribute to biotype variability in virus transmission (Brown & Bird, 1992;Bedford et al., 1994;Markham et al., 1996), and that several factors, such as; the different rates of morphological variation in endosymbionts (Costa et al., 1995); the differences in host utilization (Brown & Bird, 1992;Bedford et al., 1994a); the ability to induce physiological changes in some hosts (Costa & Brown, 1991;Cohen et al., 1992;Bedford et al., 1994), and the occurrence of esterase and DNA profile differences (Legg, 1994;Brown et al., 1995) are related to biotype variability.These considerations imply that B. tabaci samples should be also collected and submitted to lab analyses in addition to leaf samples for CBSD-like causal agent identification.
Data collected from deep sequencing in DR, Congo samples can be used to develop molecular diagnostic assays for the causative agents in western DR, Congo and others the central African countries (Congo Republic, Angola, Central Africa and Gabon) where CBSD-like symptoms are suspected. org

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