Elimination of Cassava Brown Streak Virus from Infected Cassava

Cassava brown streak disease (CBSD) is an economically important disease of cassava (Manihot esculenta crantz) caused by Cassava brown streak virus (CBSV). Use of clean planting material is one of the strategies for disease management. However, obtaining clean planting material for some farmer-preferred varieties is often difficult. This research was aimed at evaluating the effect of meristem tip sizes, effects of varying concentration levels of ribavirin and salicylic acid and determining the efficacy of thermotherapy in combination with either meristem tip culture or chemotherapy in the elimination of CBSV from infected cassava. CBSV infected cuttings of Guzo variety collected from Coast province of Kenya were established and maintained in a greenhouse at the Plant Quarantine Station in Kenya Plant Health Inspectorate Service in Muguga were used as test plants. Cassava leaves were sampled from eighteen cassava plants of Guzo variety and virus indexing was done using Reverse Transcriptase-Polymerase Chain Reaction with virus specific primers and those that tested positive for CBSV were used as initiation materials. From the in vitro plantlets established, the second sub-cultures were subjected to the virus elimination procedures. In vitro meristems (0.5mm, 1mm, 2mm and 10mm) were obtained and cultured in modified Murashige and Skoog media. For chemotherapy, nodes were cultured in MS media supplemented with antivirals at 0mg/l, 10mg/l, 20mg/l, 30mg/l. In the combination treatments single nodal plantlets were subjected to thermotherapy at 38ºC for twenty one days then excised meristem tips (1.0mm) with some plants being subjected to ribavirn treatments at (10mg/l, 20mg/l and 30mg/l). Data was analysed using Genstat 13th edition (2013). The regeneration of plants established from 0.5mm was 63% while 2mm was 88%. In chemotherapy survival of shoots was observed to decrease with increase in the antiviral concentrations. Ribavirin at 10mg/l recorded the highest rate of survival compared to the other treatments. On the other hand salicylic acid exhibited the least survival rate compared to ribavirin. The number of plants testing negative was observed to increase with increase in concentration for both chemicals. At 30 mg/l of ribavirin and salicylic; 88.8% and 100% of virus free plantlets were produced respectively. Thermotherapy (38°C) combined with meristem tips (1mm) resulted in 68% of regenerated plants with 84% being virus free. Invitro plants that had been thermo treated and then subjected to chemotherapy did not give the expected results since all plants died. Thermotherapy at (38°C) for a period of twenty one days combined with meristem tip culture can be used for production of virus free cassava. Keywords: Cassava brown streak virus, thermotherapy, chemotherapy, meristem tip culture, Virus elimination

Identification and analysis of cassava genotype TME3 bacteria artificial chromosome libraries for characterization of the cassava mosaic disease

Cassava is an economically important crop in sub-Saharan Africa; however, its yield potential is constrained by cassava mosaic disease (CMD) infection. Classical genetics and biotechnology are being harnessed to overcome the disease and secure yields for farmers. The CMD2 resistance locus flanked by three simple sequence repeats (SSR) markers and one sequence characterized amplified region (SCAR) marker were mapped in West African genotypes and shown to impart qualitative resistant to all species of CMGs. However, gene(s) associated with the CMD2 locus and their mode of actions remains unknown. In an effort to discover gene(s) located in CMD2 locus region, TME3 BAC collections were screened for the presence of CMD2 flanking markers. CMD susceptible and resistant cassava genotypes were found to contain 100% of the markers flanking CMD2 locus. SNPs and nucleotide deletions were identified within the marker sequences but there was no evidence of trait and marker association. All the SSR markers flanking CMD2, and the more recently characterized CMD3 loci were to be located on chromosome 12. Through BAC pools library hybridization with marker probes, 130 BACs were identified, but only 23 BACs contained at least CMD2 specific two markers. Whole BAC sequencing identified five clones that mapped to the marker regions. BAC29 assembled into a 100 kb contig and encoded tandem repeats of three full length R genes (3.5 kb) and two partial repeats. These R genes were conserved and highly expressed in CMD susceptible and CMD resistant cassava genotypes. Promoter sequences derived from R genes showed similar transient expression of GUS as 35S promoter. On cassava genome V6.1 BAC29 sequences were mapped to chromosome 16, eliminating their potential role in CMD resistant.Keywords: Bacteria artificial chromosome, CMD2, cassava, cassava mosaic diseas

Weed species in tomato production and their role as alternate hosts of 'Tomato spotted wilt virus' and its vector 'Frankliniella occidentalis'

'Tomato spotted wilt virus' (TSWV) is an important plant virus that infects a wide range of hosts including weeds making its management difficult. A survey was undertaken to establish the occurrence of weed species in tomato production systems in Kenya and their role as hosts of TSWV and its vectors. Selected weed species were further evaluated for their reaction to TSWV, transmission efficiency by 'Frankliniella occidentalis' and ability to support thrips reproduction. Of the 43 weed species identified in the field, 29 species had been reported as hosts of TSWV, twowere non-hosts and 11 had no record of their status. Among the more common species, 'Amaranthus hybridus', 'Solanum nigrum', 'Tagetes minuta' and 'Datura stramonium' were susceptible to the virus and supported high levels of thrips reproduction. The TSWV could not be transmitted to 'Galinsoga parviflora' and 'Sonchus oleraceus' by 'F. occidentalis' despite them being highly susceptible in mechanical transmission tests. There was a significant correlation between feeding damage and number of larvae of 'F. occidentalis' on different weeds. Occurrence of weeds that support thrips reproduction and are good hosts of TSWV is a clear indicator of their role in epidemiology and the importance of their management for disease control

Effect of bio-stimulators on Growth and Yield of Cowpea leaves (Vigna unguiculata Walp) produced in Juja and Katumani in Kenya

Bio-stimulators are organic products that have been found to enhance crop growth and productivity through mitigating against biotic and abiotic stresses. Four Cowpea varieties namely K80, M66, KVU 27-1 and Kenya Kunde; commercially released & adopted by local households for vegetable production were used to evaluate the impact of bio-stimulators on growth and leaf yield of cowpea in Juja and Katumani from May to July, 2013. The treatments were arranged in a factorial structure, laid out in an RCBD, which were combination of 9 levels of bio-stimulators, with the 4 cowpea varieties planted per site, and giving a total of 36 treatments for each site replicated 3 times. The plots measured 2.5m by 2.5m with spacing of 60cm by 20cm. Parameters under investigation included germination rate, days to germination, plant height, and number of leaves produced and number of nodules formed. Cultural practices such as weeding and pest control were done as need arose. The results showed that there were significant differences in days to germination, germination rate, plant population, chlorophyll levels, leaf weight, plant height and leaf area but not in leaf area between Juja and Katumani. Earliest germination and highest leaf weight were in Katumai whereas highest plant population, germination rate and chlorophyll levels and leaf area were in Juja. Humates improved germination rate, plant height, leaf weight and nodule development in Juja while seaweed extracts improved days to germination, germination rate, plant height, leaf yield, leaf weight and nodule formation in Katumani. Interaction between site and bio-stimulators was recorded in germination rate, days to germination, plant population and in number of leaves produced. Therefore bio-stimulators should be adopted in production of cowpea leaves particularly offseason for food & nutritional security and increasing household incomes in Keny

A metagenomic study of DNA viruses from samples of local varieties of common bean in Kenya

Common bean (Phaseolus vulgaris L.) is the primary source of protein and nutrients in the majority of households in sub-Saharan Africa. However, pests and viral diseases are key drivers in the reduction of bean production. To date, the majority of viruses reported in beans have been RNA viruses. In this study, we carried out a viral metagenomic analysis on virus symptomatic bean plants. Our virus detection pipeline identified three viral fragments of the double-stranded DNA virus Pelargonium vein banding virus (PVBV) (family, Caulimoviridae, genus Badnavirus). This is the first report of the dsDNA virus and specifically PVBV in legumes to our knowledge. In addition two previously reported +ssRNA viruses the bean common mosaic necrosis virus (BCMNVA) (Potyviridae) and aphid lethal paralysis virus (ALPV) (Dicistroviridae) were identified. Bayesian phylogenetic analysis of the Badnavirus (PVBV) using amino acid sequences of the RT/RNA-dependent DNA polymerase region showed the Kenyan sequence (SRF019_MK014483) was closely matched with two Badnavirus viruses: Dracaena mottle virus (DrMV) (YP_610965) and Lucky bamboo bacilliform virus (ABR01170). Phylogenetic analysis of BCMNVA was based on amino acid sequences of the Nib region. The BCMNVA phylogenetic tree resolved two clades identified as clade (I and II). Sequence from this study SRF35_MK014482, clustered within clade I with other Kenyan sequences. Conversely, Bayesian phylogenetic analysis of ALPV was based on nucleotide sequences of the hypothetical protein gene 1 and 2. Three main clades were resolved and identified as clades I–III. The Kenyan sequence from this study (SRF35_MK014481) clustered within clade II, and nested within a sub-clade; comprising of sequences from China and an earlier ALPV sequences from Kenya isolated from maize (MF458892). Our findings support the use of viral metagenomics to reveal the nascent viruses, their viral diversity and evolutionary history of these viruses. The detection of ALPV and PVBV indicate that these viruses have likely been underreported due to the unavailability of diagnostic tools

Metagenomic analysis of plant viruses associated with papaya ringspot disease in Carica papaya L. in Kenya

Carica papaya L. is an important fruit crop grown by small- and large-scale farmers in Kenya for local and export markets. However, its production is constrained by papaya ringspot disease (PRSD). The disease is believed to be caused by papaya ringspot virus (PRSV). Previous attempts to detect PRSV in papaya plants showing PRSD symptoms, using enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) procedures with primers specific to PRSV, have not yielded conclusive results. Therefore, the nature of viruses responsible for PRSD was elucidated in papaya leaves collected from 22 counties through Illumina MiSeq next-generation sequencing (NGS) and validated by RT-PCR and Sanger sequencing. Viruses were detected in 38 out of the 48 leaf samples sequenced. Sequence analysis revealed the presence of four viruses: a Potyvirus named Moroccan watermelon mosaic virus (MWMV) and three viruses belonging to the genus Carlavirus. The Carlaviruses include cowpea mild mottle virus (CpMMV) and two putative Carlaviruses —closely related but distinct from cucumber vein-clearing virus (CuVCV) with amino acid and nucleotide sequence identities of 75.7–78.1 and 63.6–67.6%, respectively, in the coat protein genes. In reference to typical symptoms observed in the infected plants, the two putative Carlaviruses were named papaya mottle-associated virus (PaMV) and papaya mild mottle-associated virus (PaMMV). Surprisingly, and in contrast to previous studies conducted in other parts of world, PRSV was not detected. The majority of the viruses were detected as single viral infections, while a few were found to be infecting alongside another virus (for example, MWMV and PaMV). Furthermore, the NGS and RT-PCR analysis identified MWMV as being strongly associated with ringspot symptoms in infected papaya fruits. This study has provided the first complete genome sequences of these viruses isolated from papaya in Kenya, together with primers for their detection—thus proving to be an important step towards the design of long-term, sustainable disease management strategies

Evolutionary insights of Bean common mosaic necrosis virus and Cowpea aphid-borne mosaic virus

Plant viral diseases are one of the major limitations in legume production within sub-Saharan Africa (SSA), as they account for up to 100% in production losses within smallholder farms. In this study, field surveys were conducted in the western highlands of Kenya with viral symptomatic leaf samples collected. Subsequently, next-generation sequencing was carried out to gain insights into the molecular evolution and evolutionary relationships of Bean common mosaic necrosis virus (BCMNV) and Cowpea aphid-borne mosaic virus (CABMV) present within symptomatic common bean and cowpea. Eleven near-complete genomes of BCMNV and two for CABMV were obtained from western Kenya. Bayesian phylogenomic analysis and tests for differential selection pressure within sites and across tree branches of the viral genomes were carried out. Three well–supported clades in BCMNV and one supported clade for CABMNV were resolved and in agreement with individual gene trees. Selection pressure analysis within sites and across phylogenetic branches suggested both viruses were evolving independently, but under strong purifying selection, with a slow evolutionary rate. These findings provide valuable insights on the evolution of BCMNV and CABMV genomes and their relationship to other viral genomes globally. The results will contribute greatly to the knowledge gap involving the phylogenomic relationship of these viruses, particularly for CABMV, for which there are few genome sequences available, and inform the current breeding efforts towards resistance for BCMNV and CABMV

Metagenomic analyses and genetic diversity of Tomato leaf curl Arusha virus affecting tomato plants in Kenya.

Funder: Bill and Melinda Gates Foundation; doi: http://dx.doi.org/10.13039/100000865BACKGROUND: Tomato production is threatened worldwide by the occurrence of begomoviruses which are associated with tomato leaf curl diseases. There is little information on the molecular properties of tomato begomoviruses in Kenya, hence we investigated the population and genetic diversity of begomoviruses associated with tomato leaf curl in Kenya. METHODS: Tomato leaf samples with virus-like symptoms were obtained from farmers' field across the country in 2018 and Illumina sequencing undertaken to determine the genetic diversity of associated begomoviruses. Additionally, the occurrence of selection pressure and recombinant isolates within the population were also evaluated. RESULTS: Twelve complete begomovirus genomes were obtained from our samples with an average coverage of 99.9%. The sequences showed 95.7-99.7% identity among each other and 95.9-98.9% similarities with a Tomato leaf curl virus Arusha virus (ToLCArV) isolate from Tanzania. Analysis of amino acid sequences showed the highest identities in the regions coding for the coat protein gene (98.5-100%) within the isolates, and 97.1-100% identity with the C4 gene of ToLCArV. Phylogenetic algorithms clustered all Kenyan isolates in the same clades with ToLCArV, thus confirming the isolates to be a variant of the virus. There was no evidence of recombination within our isolates. Estimation of selection pressure within the virus population revealed the occurrence of negative or purifying selection in five out of the six coding regions of the sequences. CONCLUSIONS: The begomovirus associated with tomato leaf curl diseases of tomato in Kenya is a variant of ToLCArV, possibly originating from Tanzania. There is low genetic diversity within the virus population and this information is useful in the development of appropriate management strategies for the disease in the country

The process and lessons of exchanging and managing in-vitro elite germplasm to combat CBSD and CMD in Eastern and Southern Africa

Varieties with resistance to both cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) can reverse food and income security threats affecting the rural poor in Eastern and Southern Africa. The International Institute of Tropical Agriculture is leading a partnership of five national (Malawi, Mozambique, Kenya, Tanzania and Uganda) cassava breeding programs to exchange the most elite germplasm resistant to both CMD and CBSD. This poster documents the process and the key learning lessons. Twenty to 25 stem cuttings of 31 clones comprising of 25 elite clones (5 per country), two standard checks (Kibandameno from Kenya and Albert from Tanzania), and four national checks (Kiroba and Mkombozi from Tanzania, Mbundumali from Malawi, and Tomo from Mozambique) were cleaned and indexed for cassava viruses at both the Natural Resources Institute in the United Kingdom and Kenya Plant Health Inspectorate Services, in Kenya. About 75 in-vitro plantlets per clone were sent to Genetic Technologies International Limited, a private tissue culture lab in Kenya, and micro-propagated to ≥1500 plantlets. Formal procedures of material transfer between countries including agreements, import permission and phytosanitary certification were all ensured for germplasm exchange. At least 300 plantlets of each elite and standard check clones were sent to all partner countries, while the national checks were only sent to their respective countries of origin. In each country, the in-vitro plantlets were acclimatized under screen house conditions and transplanted for field multiplication as a basis for multi-site testing. Except for Tomo, a susceptible clone, all the clones were cleaned of the viruses. However, there was varied response to the cleaning process between clones, e.g. FN-19NL, NASE1 and Kibandameno responded slowly. Also, clones responded differently to micro-propagation protocols at GTIL, e.g. Pwani, Tajirika, NASE1, TME204 and Okhumelela responded slowly. Materials are currently being bulked at low disease pressure field sites in preparation for planting at 5-8 evaluation sites per country. The process of cleaning, tissue culture mass propagation, exchange and local hardening off/bulking has been successful for the majority of target varieties. Two key lessons derived from the process are that adequate preparations of infrastructure and trained personnel are required to manage the task, and that a small proportion of varieties are recalcitrant to tissue culture propagation

Distribution, charactisation and management of 'Tomato spotted wilt virus' and its vectors in tomato production systems in Kenya

'Tomato spotted wilt virus' (family 'Bunyaviridae', genus 'Tospovirus') is an important virus infecting horticultural crops and is transmitted solely by thrips species. The virus infects a wide range of plant species and presence of 'Frankliniella occidentalis', reported to be the most efficient vector species has complicated its management. TSWV was first reported in Kenya in 1999, causing serious yield losses in tomato production, and has continued to cause sporadic yield losses. This study was undertaken to determine the distribution and genetic diversity of the virus a decade after its incursion into Kenya. The study further evaluated important factors in the epidemiology of the disease and its management