Molecular Characterization of Viruses Causing the Cassava Brown Streak Disease Epidemic in Eastern Africa

Cassava brown streak disease (CBSD) was described for the first time in Tanganyika (now Tanzania) about seven decades ago. Tanganyika (now Tanzania) about seven decades ago. It was endemic in the lowland areas of East Africa and inland parts of Malawi and caused by Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae). However, in 1990s CBSD was observed at high altitude areas in Uganda. The causes for spread to new locations were not known.The present work was thus initiated to generate information on genetic variability, clarify the taxonomy of the virus or viruses associated with CBSD in Eastern Africa as well as to understand the evolutionary forces acting on their genes. It also sought to develop a molecular based diagnostic tool for detection of CBSD-associated virus isolates. Comparison of the CP-encoding sequences of CBSD-associated virus isolates collected from Uganda and north-western Tanzania in 2007 and the partial sequences available in Genbank revealed occurrence of two genetically distinct groups of isolates. Two isolates were selected to represent the two groups. The complete genomes of isolates MLB3 (TZ:Mlb3:07) and Kor6 (TZ:Kor6:08) obtained from North-Western (Kagera) and North-Eastern (Tanga) Tanzania, respectively, were sequenced. The genomes were 9069 and 8995 nucleotides (nt), respectively. They translated into polyproteins that were predicted to yield ten mature proteins after cleavage. Nine proteins were typical in the family Potyviridae, namely P1, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP, but the viruses did not contain HC-Pro. Interestingly, genomes of both isolates contained a Maf/HAM1-like sequence (HAM1h; 678 nucleotides, 25 kDa) recombined between the NIb and CP domains in the 3’-proximal part of the genomes. HAM1h was also identified in Euphorbia ringspot virus (EuRSV) whose sequence was in GenBank. The HAM1 gene is widely spread in both prokaryotes and eukaryotes. In yeast (Saccharomyces cerevisiae) it is known to be a nucleoside triphosphate (NTP) pyrophosphatase. Novel information was obtained on the structural variation at the N-termini of polyproteins of viruses in the genus Ipomovirus. Cucumber vein yellowing virus (CVYV) and Squash vein yellowing virus (SqVYV) contain a duplicated P1 (P1a and P1b) but lack the HC-Pro. On the other hand, Sweet potato mild mottle virus (SPMMV), has a single but large P1 and has HC-Pro. Both virus isolates (TZ:Mlb3:07 & TZ:Kor6:08) characterized in this study contained a single P1 and lacked the HC-Pro which indicates unique evolution in the family Potyviridae. Comparison of 12 complete genomes of CBSD-associated viruses which included two genomes characterized in this study, revealed genetic identity of 69.0–70.3% (nt) and amino acid (aa) identities of 73.6–74.4% at polyprotein level. Comparison was also made among 68 complete CP sequences, which indicated 69.0-70.3 and 73.6-74.4 % identity at nt and aa levels, respectively. The genetic variation was large enough for dermacation of CBSD-associated virus isolates into two distinct species. The name CBSV was retained for isolates that were related to CBSV isolates available in database whereas the new virus described for the first time in this study was named Ugandan cassava brown streak virus (UCBSV) by the International Committee on Virus Taxonomy (ICTV). The isolates TZ:Mlb3:07 and TZ:Kor6:08 belong to UCBSV and CBSV, respectively. The isolates of CBSV and UCBSV were 79.3-95.5% and 86.3-99.3 % identitical at nt level, respectively, suggesting more variation amongst CBSV isolates. The main sources of variation in plant viruses are mutations and recombination. Signals for recombination events were detected in 50% of isolates of each virus. Recombination events were detected in coding and non-coding (3’-UTR) sequences except in the 5’UTR and P3. There was no evidence for recombination between isolates of CBSV and UCBSV. The non-synonomous (dN) to synonomous (dS) nucleotide substitution ratio (ω) for the HAM1h and CP domains of both viruses were ≤ 0.184 suggesting that most sites of these proteins were evolving under strong purifying selection. However, there were individual amino acid sites that were submitted to adaptive evolution. For instance, adaptive evolution was detected in the HAM1h of UCBSV (n=15) where 12 aa sites were under positive selection (P< 0.05) but not in CBSV (n=12). The CP of CBSV (n=23) contained 12 aa sites (p<0.01) while only 5 aa sites in the CP gene of UCBSV were predicted to be submitted to positive selection pressure (p<0.01). The advantages offered by the aa sites under positive selection could not be established but occurrence of such sites in the terminal ends of UCBSV-HAMIh, for example, was interpreted as a requirement for proteolysis during polyprotein processing. Two different primer pairs that simultaneously detect UCBSV and CBSV isolates were developed in this study. They were used successfully to study distribution of CBSV, UCBSV and their mixed infections in Tanzania and Uganda. It was established that the two viruses co-infect cassava and that incidences of co-infection could be as high as 50% around Lake Victoria on the Tanzanian side. Furthermore, it was revealed for the first time that both UCBSV and CBSV were widely distributed in Eastern Africa. The primer pair was also used to confirm infection in a close relative of cassava, Manihot glaziovii (Müller Arg.) with CBSV. DNA barcoding of M. glaziovii was done by sequencing the matK gene. Two out of seven M. glaziovii from the coastal areas of Korogwe and Kibaha in north eastern Tanzania were shown to be infected by CBSV but not UCBSV isolates. Detection in M. glaziovii has an implication in control and management of CBSD as it is likely to serve as virus reservoir. This study has contributed to the understanding of evolution of CBSV and UCBSV, which cause CBSD epidemic in Eastern Africa. The detection tools developed in this work will be useful in plant breeding, verification of the phytosanitary status of materials in regional and international movement of germplasm, and in all diagnostic activities related to management of CBSD. Whereas there are still many issues to be resolved such as the function and biological significance of HAM1h and its origin, this work has laid a foundation upon which the studies on these aspects can be based.Kassava on trooppisten alueiden merkittävimpiä ruokaturvakasveja. Kassavan ruskoviirutauti on levinnyt 2000-luvulla Intian valtameren rannikkoalueilta sisämaan ja ylänköjen kassavaviljelmille Itä-Afrikassa. Tauti pilaa ravinnoksi käytettävät varastojuuret aiheuttaen niissä kuivamätää. Tässä tutkimuksessa havaittiin, että tauti liittyy kahden toisilleen lähisukuisen viruksen tartuntaan. Taudin aiheuttajina toimivien ipomovirusten genomien havaittiin olevan rakenteeltaan poikkeuksellisia muihin Potyviridae-ryhmään kuuluviin viruksiin verrattuna. Lisäksi virukset sisältävät todennäköisesti kasvisolusta kaapatun geenin. Sen toiminta saattaa liittyä viruksen tarpeeseen suojata genominsa hajotukselta kasveissa, jotka kärsivät esimerkiksi kuumuuden aiheuttamasta stressistä. Toinen viruksista havaittiin kassavalle lähisukuisessa, villinä kasvavassa lajissa, joka sekin on tuotu Afrikkaan Etelä-Amerikasta. Ruskoviirutautia aiheuttavien virusten diagnostiikkaa varten kehitettiin PCR-testi, joka tunnistaa molemmat virukset samanaikaisesti, mutta pystyy myös erottelemaan ne. Testimenetelmän avulla virusten levinneisyyttä pystyttiin kartoittamaan aiempaa laajemmin. Molemmat virukset ja niiden sekainfektiot kassavassa havaittiin yleisiksi niin Ugandan kuin Tansaniankin viljelmillä. Tulokset tuottivat uutta tietoa virusten evoluutiosta. Tulokset edistävät myös merkittävästi kassavan ruskoviirutaudin leviämisen estämiseen tarvittavaa kasvintarkastustoimintaa, sillä toinen viruksista oli aiemmin tuntematon eikä kummallekaan virukselle ole ollut helppokäyttöistä testausmenetelmää

Development of a molecular-based detection tool for sweet potato leaf curl viruses and determination of their incidence levels in Tanzania

Sweet potato (Ipomoea batatas L.) is an important food security crop in sub-Saharan Africa, where its production is constrained by devastating diseases; including those caused by sweet potato leaf curl viruses (SPLCVs; Begomovirus; Geminiviridae). The objective of this study was to develop a molecular-based diagnostic detection tool for SPLCVs, as well as to generate information on their geographical distribution and incidence in Tanzania. A comprehensive survey of SPLCVs was done in all major sweet potato growing areas in Tanzania. Incidences of SPLCVs and their causative diseases were determined visually and by polymerase chain reaction (PCR), using primers designed, optimised and validated. DNA was extracted from 4166 sweet potato leaf samples and detection of SPLCVs was done. Visual incidence of disease symptoms ranged from 0 to 100%; while PCR-based incidence of SPLCVs ranged from 0 to 60%. The highest mean PCR-based incidence of SPLCVs was 32%. SPLCVs occurred in all sweet potato growing areas. There was a negative correlation between the visually assessed incidence of disease symptoms and PCR-based incidence of SPCLVs (r = -0.122 and R2 = 0.012). A weak positive correlation between altitude and PCR-based incidence of SPLCVs was also found. In ten-fold serially diluted sweet potato DNA samples, using our new primer pair d1-SPLCVF/d1-SPLCVR, the detection limit of SPLCVs was at the dilution of 10-3. The youngest fully expanded leaf of the sweet potato plant was the best for PCR detection of SPLCVs. These findings will be useful for strategic deployment of planting material and conducting sweet potato breeding experiments for resistance against SPLCVs. &nbsp

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

Survey of Potato aphid (Myzus persicae) from Selected on-farm Potato growing Areas in Mbeya and Njombe regions of Tanzania

Potato aphid green peach (Myzus persicae Sulzer) is one of the serious pests in potato (Solanum tuberosum) production areas in the world. A survey was carried out during 2014 potato growing season to assess aphid and Coccinellid population abundance in potato production areas in Mbeya and Njombe regions situated in southern highlands of Tanzania. Aphid and Coccinellid abundance were sampled from 30 leaves selected randomly from ten plants. Estimates of aphid abundance from potato fields were carried out from transectline replicated three times. A total of 1,370 aphid populations were sampled during the survey period. Abundance from young leaves was 898, followed by medium leaves 270 and the least was old leaves represented by 202 individuals. The relative abundance of aphid population resulted from 15 minutes sampling effort per transect ranked from 8 individuals from Swaya village to 171 individuals from Atwelo village and the rest were in between. The differences between selected plant parts showed significant differences (p = 0.001). The results from this survey provides database on aphid population and Coccinellid natural enemies of potato aphid in potato growing areas which can be used for future research in the selected regions. Key words: Myzus persicae, potato aphid, Coccinelli

Endemism and reemergence potential of the ipomovirus Sweet potato mild mottle virus (family Potyviridae) in Eastern Africa: half a century of mystery

Viruses have the ability to frequently colonize new hosts and ecological niches because of their inherently high genetic and evolutionary plasticity. However, a virus may emerge and remain of no or less economic importance until changes in viral and/or environmental factors dictate its epidemiological status. An example is sweet potato mild mottle virus (SPMMV), which was first reported in the 1970s on sweetpotatoes in eastern Africa, has remained endemic in the region and poorly understood, yet accounting for 60-95% losses especially in mixed infections. Unlike other sweetpotato viruses which have a global incidence, SPMMV has never been confirmed outside eastern Africa. This implicates the region as its center of origin, but does not fully account for SPMMV’s exclusive geographic delimitation to eastern Africa. Despite its importance, several mysteries and research gaps surround SPMMV, which decelerate efforts for effective virus disease management in sweetpotato. The aim of this review is to articulate research gaps, propose pivotal scientific directions and stimulate knowledge generation for better management of virus diseases in sweetpotato. Vector-mediated transmission of SPMMV remains enigmatic. Here we postulate testable hypotheses to explain SPMMV transmission. Comparisons between SPMMV and cassava brown streak ipomoviruses demonstrate epidemiological “hallmarks” for monitoring SPMMV. Evolutionary forces on SPMMV coupled with the virus’ broad host range imply a ‘silent build up’ of better fit variants in a changing climate, and this could explode into a worse disease conundrum. These information gaps need urgent filling to ease future management of virus disease emergences in sweetpotato

Pathogenic seedborne viruses are rare but Phaseolus vulgaris endornaviruses are common in bean varieties grown in Nicaragua and Tanzania

Common bean (Phaseolus vulgaris) is an annual grain legume that was domesticated in Mesoamerica (Central America) and the Andes. It is currently grown widely also on other continents including Africa. We surveyed seedborne viruses in new common bean varieties introduced to Nicaragua (Central America) and in landraces and improved varieties grown in Tanzania (eastern Africa). Bean seeds, harvested from Nicaragua and Tanzania, were grown in insect-controlled greenhouse or screenhouse, respectively, to obtain leaf material for virus testing. Equal amounts of total RNA from different samples were pooled (30-36 samples per pool), and small RNAs were deep-sequenced (Illumina). Assembly of the reads (21-24 nt) to contiguous sequences and searches for homologous viral sequences in data-bases revealed Phaseolus vulgaris endornavirus 1 (PvEV-1) and PvEV-2 in the bean varieties in Nicaragua and Tanzania. These viruses are not known to cause symptoms in common bean and are considered non-pathogenic. The small-RNA reads from each pool of samples were mapped to the previously characterized complete PvEV-1 and PvEV-2 sequences (genome lengths ca. 14 kb and 15 kb, respectively). Coverage of the viral genomes was 87.9-99.9%, depending on the pool. Coverage per nucleotide ranged from 5 to 471, confirming virus identification. PvEV-1 and PvEV-2 are known to occur in Phaseolus spp. in Central America, but there is little previous information about their occurrence in Nicaragua, and no information about occurrence in Africa. Aside from Cowpea mild mosaic virus detected in bean plants grown from been seeds harvested from one region in Tanzania, no other pathogenic seedborne viruses were detected. The low incidence of infections caused by pathogenic viruses transmitted via bean seeds may be attributable to new, virus-resistant CB varieties released by breeding programs in Nicaragua and Tanzania.Peer reviewe

Host and virus effects on reversion in cassava affected by cassava brown streak disease

The phenomenon of virus-infected plants naturally recovering health is known as ‘reversion’, and is a type of resistance mechanism exploited in some crop plants for disease control. Various parameters were investigated that affect reversion from cassava brown streak disease (CBSD) in three cassava varieties (Albert, Kaleso and Kiroba) that differ in levels of resistance to the disease. Cassava plants were inoculated by grafting with two virus species (Ugandan cassava brown streak virus, UCBSV and Cassava brown streak virus, CBSV) that cause CBSD, and the plants grown from them were subsequently assessed for reversion. The rate of reversion depended on the cassava variety, virus species, and the length and position of the stem cuttings used. A significantly high proportion of progenies were virus-free (reverted) for the resistant variety Kaleso (64·1% for UCBSV and 54·9% of CBSV), compared to the tolerant variety Kiroba (56·7 and 45·5%) and the susceptible control Albert (38·9 and 35·1%). The highest number of virus-free plants was generated from short 10 cm long cuttings (e.g. 60·1% for Kaleso for CBSV) compared to 20 cm long stem cuttings (e.g. 21·4% for Albert). Cuttings taken from upper stems of diseased plants produced most virus-free progenies compared to middle and lower parts. More than 50% virus-free plants were obtained in the resistant and tolerant varieties. This is a highly valuable finding and could be exploited for developing strategies to control the current CBSD epidemic in eastern and central Africa

Cassava brown streak disease: historical timeline, current knowledge and future prospects

Cassava is the second most important staple food crop in terms of per capita calories consumed in Africa and holds potential for climate change adaptation. Unfortunately, productivity in East and Central Africa is severely constrained by two viral diseases: cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). CBSD was first reported in 1936 from northeast Tanzania. For approximately seventy years CBSD was restricted to coastal East Africa and so had a relatively low impact on food security compared to CMD. However, at the turn of the 21st century CBSD re-emerged further inland, in areas around Lake Victoria and it has since spread through many East and Central African countries, causing high yield losses and jeopardising the food security of subsistence farmers. This recent re-emergence has attracted intense scientific interest, with studies shedding light on CBSD viral epidemiology, sequence diversity, host interactions and potential sources of resistance within the cassava genome. This review reflects on 80 years of CBSD research history (1936 – 2016) with a timeline of key events. We provide insights into current CBSD knowledge, management efforts and future prospects for improved understanding needed to underpin effective control and mitigation of impacts on food security

Strategies for the construction of cassava brown streak disease viral infectious clones

Cassava brown streak disease (CBSD) has major impacts on yield and quality of the tuberous roots of cassava in Eastern and Central Arica. At least two Potyviridae species cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Cloned viral genome sequences known as infectious clones (ICs) have been important in the study of other viruses, both as a means of standardising infectious material and characterising viral gene function. IC construction is often technically challenging for Potyviridae due to sequence instability in E. coli. Here, we evaluate three methods for the construction of infectious clones for CBSD. Whilst a simple IC for in vitro transcription was made for UCBSV isolate ‘Kikombe’, such an approach failed to deliver full-length clones for CBSV isolates ‘Nampula’ or ‘Tanza’, necessitating more complex approaches for their construction. The ICs successfully generated symptomatic infection in the model host N. benthamiana and in the natural host cassava. This shows that whilst generating ICs for CBSV is still a technical challenge, a structured approach, evaluating both in vitro and in planta transcription systems should successfully deliver ICs, allowing further study into the symptomology and virulence factors in this important disease complex

Amplicon sequencing identified a putative pathogen, Macrophomina phaseolina, causing wilt in African eggplant (Solanum aethiopicum) grown in Tanzania and Uganda

African eggplant (Solanum aethiopicum L.) is one of the most common traditional vegetables in Tanzania and Uganda, but its productivity is severely affected by wilt diseases caused by a number of pathogens. Plant stem and root samples were collected in several fields from many neighboring diseased and healthy plants of the Gilo group in Tanzania and from the Shum group in Uganda to identify putative pathogens causing wilt on African eggplants. Through amplicon sequencing of sampled diseased and healthy tissues, we identified putative causal pathogens for the wilt symptoms. Wilting of S. aethiopicum in Uganda is most likely caused by the bacterial pathogen Ralstonia solanacearum whereas, in Tanzania, wilt is most likely caused by the fungal pathogen Macrophomina phaseolina, infecting roots. Infection of stems by Fusarium solani may also contribute to the wilt symptoms in Tanzania. Further artificial inoculation under controlled conditions confirmed that M. phaseolina can cause typical wilting symptoms on S. aethiopcium genotypes. The discovery of different putative causal agents of wilt in the crop demonstrates the need for site specific etiological analysis of wilt before developing and implementing effective control methods. Further research is needed to confirm the results and develop appropriate management measures against specific wilt pathogens