Assessing the effect of sample storage time on viral detection using a rapid and cost-effective CTAB-based extraction method.

BACKGROUND: Cassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce. RESULTS: The effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.-vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p < 0.005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods. CONCLUSION: Our method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions.

Cassava brown streak virus evolves with a nucleotide-substitution rate that is typical for the family Potyviridae.

The ipomoviruses (family Potyviridae) that cause cassava brown streak disease (cassava brown streak virus [CBSV] and Uganda cassava brown streak virus [UCBSV]) are damaging plant pathogens that affect the sustainability of cassava production in East and Central Africa. However, little is known about the rate at which the viruses evolve and when they emerged in Africa - which inform how easily these viruses can host shift and resist RNAi approaches for control. We present here the rates of evolution determined from the coat protein gene (CP) of CBSV (Temporal signal in a UCBSV dataset was not sufficient for comparable analysis). Our BEAST analysis estimated the CBSV CP evolves at a mean rate of 1.43 × 10-3 nucleotide substitutions per site per year, with the most recent common ancestor of sampled CBSV isolates existing in 1944 (95% HPD, between years 1922 - 1963). We compared the published measured and estimated rates of evolution of CPs from ten families of plant viruses and showed that CBSV is an average-evolving potyvirid, but that members of Potyviridae evolve more quickly than members of Virgaviridae and the single representatives of Betaflexiviridae, Bunyaviridae, Caulimoviridae and Closteroviridae.

Characterization of phenotypic traits associated with anthracnose resistance in selected common bean (Phaseolus vulgaris L.) breeding material.

Anthracnose caused by Colletotrichum lindemuthianum is the major common bean disease worldwide causing complete yield loss under favourable disease conditions. This study aimed to determine phenotypic traits associated with anthracnose resistance for future use in breeding programmes. Twenty-two common bean varieties (CBVs) were selected basing on susceptibility to anthracnose, advanced breeding lines, improved variety resembling advanced breeding lines and the farmer variety widely grown in Tanzania. Selected varieties were planted in anthracnose hotspot fields and the same CBVs were planted in a screen house to validate resistance to anthracnose. Anthracnose infection score, leaf length, leaf width, length of fifth internode, length of petiole, plant vigour, canopy height and canopy width were recorded. Data on number of plants emerging; days to flowering; days to maturity; plant stands at harvest; and grain yield were also collected and analysed using R software. Phenotypic traits evaluated differed significantly among genotypes, environment and genotype by environment interaction. Seventy-five percent of phenotypic traits evaluated were positively correlated to anthracnose resistance. Highly-strong correlations to anthracnose were observed on number of days to maturity, plant stands at harvest, plant vigour and grain yield. Leaf length, leaf width, length of fifth internode, length of petiole and number of stands emerging were strongly correlated to anthracnose resistance. Additive main effects and multiplicative interaction analysis (AMMI) revealed highest contribution of environment on anthracnose infection-58.9% and grain yield -84.9% compared to genotype effects on anthracnose infection -32.7% and grain yield-15.7%. Based on these results, four traits - plant vigour, number of days to maturity, number of plant stands at harvest and grain yield - are recommended for selecting anthracnose-resistant varieties. NUA 48, NUA 64 and RWR 2154 were superior varieties, resistant to anthracnose and high yielding, while Sweet Violet and VTT 923-23-10 were most stable varieties across environments. Further on-farm research is suggested to assess their performance and identify traits preferred by farmers.

SEGS-1 a cassava genomic sequence increases the severity of African cassava mosaic virus infection in Arabidopsis thaliana.

Cassava is a major crop in Sub-Saharan Africa, where it is grown primarily by smallholder farmers. Cassava production is constrained by Cassava mosaic disease (CMD), which is caused by a complex of cassava mosaic begomoviruses (CMBs). A previous study showed that SEGS-1 (sequences enhancing geminivirus symptoms), which occurs in the cassava genome and as episomes during viral infection, enhances CMD symptoms and breaks resistance in cassava. We report here that SEGS-1 also increases viral disease severity in Arabidopsis thaliana plants that are co-inoculated with African cassava mosaic virus (ACMV) and SEGS-1 sequences. Viral disease was also enhanced in Arabidopsis plants carrying a SEGS-1 transgene when inoculated with ACMV alone. Unlike cassava, no SEGS-1 episomal DNA was detected in the transgenic Arabidopsis plants during ACMV infection. Studies using Nicotiana tabacum suspension cells showed that co-transfection of SEGS-1 sequences with an ACMV replicon increases viral DNA accumulation in the absence of viral movement. Together, these results demonstrated that SEGS-1 can function in a heterologous host to increase disease severity. Moreover, SEGS-1 is active in a host genomic context, indicating that SEGS-1 episomes are not required for disease enhancement.

A New Type of Satellite Associated with Cassava Mosaic Begomoviruses.

Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated SEGS-2 (sequence enhancing geminivirus symptoms). We report here that when SEGS-2 is coinoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana, viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus-resistant Arabidopsis thaliana accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in A. thaliana. Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of an SEGS-2-encoded protein that may also be encoded by the cassava genome. IMPORTANCE Cassava is an important root crop in the developing world and a food and income crop for more than 300 million African farmers. Cassava is rising in global importance and trade as the demands for biofuels and commercial starch increase. More than half of the world's cassava is produced in Africa, where it is primarily grown by smallholder farmers, many of whom are from the poorest villages. Although cassava can grow under high temperature, drought, and poor soil conditions, its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is one of the most important viral diseases of cassava and can cause up to 100% yield losses. We provide evidence that SEGS-2, which was originally isolated from cassava crops displaying severe and atypical CMD symptoms in Tanzanian fields, is a novel begomovirus satellite that can compromise the development of durable CMD resistance.

Molecular approach to confirm traditional identification of Radopholus similis sampled in Tanzania.

Banana (Musa spp. L.) is an important staple food and cash crop for about 30% of the population in Tanzania; however, the burrowing plant-parasitic nematode Radopholus similis causes black head disease and toppling in banana plants, which results in yield losses. We collected and identified 80 specimens of R. similis from four agro-ecological zones in Tanzania using morphological characters. We then used universal and specific R. similis primers to amplify the small subunit, internal transcribed spacer and large subunit of ribosomal DNA regions of these specimens. The amplicons were subsequently sequenced and analyzed using Bayesian inference. We identified two major clades, one that comprised all R. similis sequences derived from this study and another that included R. similis and Radopholus spp. sequences obtained from GenBank, indicating the separation of this species from congeneric sequences. Our findings provide a useful, simple and rapid method for identifying burrowing nematodes. This outcome could contribute to the development of permanent, integrated pest management strategies for the control of R. similis in banana and other crops in order to reduce associated yield losses in Tanzania. To our knowledge, this is the first study of nematodes to use combined morphological and molecular methods for the identification of R. similis in Tanzania.Banana (Musa spp. L.) is an important staple food and cash crop for about 30% of the population in Tanzania; however, the burrowing plant-parasitic nematode Radopholus similis causes black head disease and toppling in banana plants, which results in yield losses. We collected and identified 80 specimens of R. similis from four agro-ecological zones in Tanzania using morphological characters. We then used universal and specific R. similis primers to amplify the small subunit, internal transcribed spacer and large subunit of ribosomal DNA regions of these specimens. The amplicons were subsequently sequenced and analyzed using Bayesian inference. We identified two major clades, one that comprised all R. similis sequences derived from this study and another that included R. similis and Radopholus spp. sequences obtained from GenBank, indicating the separation of this species from congeneric sequences. Our findings provide a useful, simple and rapid method for identifying burrowing nematodes. This outcome could contribute to the development of permanent, integrated pest management strategies for the control of R. similis in banana and other crops in order to reduce associated yield losses in Tanzania. To our knowledge, this is the first study of nematodes to use combined morphological and molecular methods for the identification of R. similis in Tanzania.

Genetic diversity and SNP's from the chloroplast coding regions of virus-infected cassava.

Cassava is a staple food crop in sub-Saharan Africa; it is a rich source of carbohydrates and proteins which currently supports livelihoods of more than 800 million people worldwide. However, its continued production is at stake due to vector-transmitted diseases such as Cassava mosaic disease and Cassava brown streak disease. Currently, the management and control of viral diseases in cassava relies mainly on virus-resistant cultivars of cassava. Thus, the discovery of new target genes for plant virus resistance is essential for the development of more cassava varieties by conventional breeding or genetic engineering. The chloroplast is a common target for plant viruses propagation and is also a potential source for discovering new resistant genes for plant breeding. Non-infected and infected cassava leaf samples were obtained from different locations of East Africa in Tanzania, Kenya and Mozambique. RNA extraction followed by cDNA library preparation and Illumina sequencing was performed. Assembling and mapping of the reads were carried out and 33 partial chloroplast genomes were obtained. Bayesian phylogenetic analysis from 55 chloroplast protein-coding genes of a dataset with 39 taxa was performed and the single nucleotide polymorphisms for the chloroplast dataset were identified. Phylogenetic analysis revealed considerable genetic diversity present in chloroplast partial genome among cultivated cassava of East Africa. The results obtained may supplement data of previously selected resistant materials and aid breeding programs to find diversity and achieve resistance for new cassava varieties.

African Basil (Ocimum gratissimum) Is a Reservoir of Divergent Begomoviruses in Uganda.

Begomoviruses are plant viruses that cause major losses to many economically important crops. Although they are poorly understood, begomoviruses infecting wild plants may have an important role as reservoirs in the epidemiology of viral diseases. This study reports the discovery and genomic characterization of three novel bipartite begomoviruses from wild and cultivated African basil (Ocimum gratissimum) plants collected in Uganda, East Africa. Based on the symptoms shown by the infected plants, the names proposed for these viruses are Ocimum yellow vein virus (OcYVV), Ocimum mosaic virus (OcMV), and Ocimum golden mosaic virus (OcGMV). Genome and phylogenetic analyses suggest that DNA-A of OcGMV is mostly related to begomoviruses infecting tomato in Africa, whereas those of OcYVV and OcMV are closely related to one another and highly divergent within the Old World begomoviruses. The DNA-A of all characterized begomovirus isolates are of a recombinant nature, revealing the role of recombination in the evolution of these begomoviruses. The viruses characterized here are the first identified in O. gratissimum and the first in Ocimum spp. in the African continent and could have important epidemiological consequences for cultivated basils and other important crops.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Expansion of the cassava brown streak pandemic in Uganda revealed by annual field survey data for 2004 to 2017.

Cassava brown streak disease (CBSD) is currently the most devastating cassava disease in eastern, central and southern Africa affecting a staple crop for over 700 million people on the continent. A major outbreak of CBSD in 2004 near Kampala rapidly spread across Uganda. In the following years, similar CBSD outbreaks were noted in countries across eastern and central Africa, and now the disease poses a threat to West Africa including Nigeria - the biggest cassava producer in the world. A comprehensive dataset with 7,627 locations, annually and consistently sampled between 2004 and 2017 was collated from historic paper and electronic records stored in Uganda. The survey comprises multiple variables including data for incidence and symptom severity of CBSD and abundance of the whitefly vector (Bemisia tabaci). This dataset provides a unique basis to characterize the epidemiology and dynamics of CBSD spread in order to inform disease surveillance and management. We also describe methods used to integrate and verify extensive field records for surveys typical of emerging epidemics in subsistence crops.

Tree Lab: Portable genomics for Early Detection of Plant Viruses and Pests in Sub-Saharan Africa.

In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall-degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This closed system delivers purified DNA with no cross-contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into nucleotide base called data locally in real-time, removing the need for high-specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and setup. To evaluate and validate capability of the system for unbiased pathogen identification by real-time sequencing in a farmer's field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or destroy cassava crops. Eight hundred (800) million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint to its production. Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus-resistant or replacement planting. This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, Internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis, including sample collection, processing and provisional sequencing results was complete in under 3 h. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation.

Immunohistochemical localization of Cassava brown streak virus and its morphological effect on cassava leaves.

The localization of Cassava brown streak virus (CBSV) in cassava (Manihot esculenta) leaf tissues was determined and cellular morphological changes in CBSV-infected tissues were evaluated. CBSV-symptomatic leaves were screened with CBSV-specific primers using reverse-transcriptase polymerase chain reaction. Immunohistochemical reactions showed precipitation in CBSV-infected but not CBSV-free tissues, demonstrating successful localization of CBSV. Microscopic inspection showed significantly larger (P < 0.001) midribs in CBSV-infected compared with control (uninfected) leaves. Viral accumulation occurred in middle and lower but rarely in young upper leaves. This immunohistochemical method for virus localization will be invaluable for efficient screening of CBSV and for breeding resistant cassava.

First report of Cassava brown streak viruses on wild plant species in Mozambique.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the main constraint to cassava (Manihot esculenta Crantz) production in Mozambique. Using RT-PCR to amplify partial coat protein nucleotide sequences, we detected for the first time the occurrence of CBSV in two non-cassava perennial wild plant species: Zanha africana (Radlk.) Exell. and Trichodesma zeylanicum (Burm.f.) R.Br., that occur widely within and near cassava fields in Nampula, Zambezia, Niassa and Cabo Delgado provinces. In addition, we also detected CBSV and UCBSV in Manihot carthaginensis subsp. glaziovii (Müell-Arg.) Allem., a wild cassava relative. These findings were verified in biological assays through mechanical inoculation of CBSV to T. zeylanicum, albeit at low rates of infection. Phylogenetic analysis clustered the CBSV isolates from the non-cassava plant species with those from cultivated cassava, with high sequence homology among CBSV (91.0-99.6%) and with UCBSV (84-92%) isolates. These results provide definitive evidence of a wider host range for CBSV and UCBSV in Mozambique, indicating that these viruses are not restricted to cultivated cassava. Our findings are key to understanding the epidemiology of CBSD and will aid in the development of sustainable management strategies for the disease.

Resistance of advanced cassava breeding clones to infection by major viruses in Uganda.

Cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) are two viral diseases that cause severe yield losses in cassava of up to 100%, thereby persistently threatening food and income security in sub-Saharan Africa. For effective management of these diseases, there is a critical need to develop and deploy varieties with dual resistance to CBSD and CMD. In this study, we determined the response of advanced breeding lines to field infection by cassava brown streak viruses (CBSVs) and cassava mosaic begomoviruses (CMBs). This aim helped in identifying superior clones for downstream breeding. In total, 220 cassava clones, three in uniform yield trials (UYTs) and 217 in a crossing block trial (CBT), were evaluated for virus and disease resistance. Field data were collected on disease incidence and severity. To detect and quantify CBSVs, 448 and 128 leaf samples from CBSD symptomatic and symptomless plants were analyzed by reverse transcription PCR and real-time quantitative PCR, respectively. In addition, 93 leaf samples from CMD symptomatic plants in the CBT were analyzed by conventional PCR using CMB species-specific primers. In the CBT, 124 (57%) cassava clones did not express CMD symptoms. Of the affected plants, 44 (55%) had single African cassava mosaic virus infection. Single Cassava brown streak virus (CBSV) infections were more prevalent (81.6%) in CBT clones than single Ugandan cassava brown streak virus (UCBSV) infection (3.2%). Of the three advanced clones in the UYT, NAROCASS 1 and NAROCASS 2 had significantly lower (P < 0.05) CBSD severity, incidence, and CBSV load than MH04/0300. In the UYT, only 22% of samples tested had CBSVs, and all showed a negative result for CMBs. The low disease incidence, severity, and viral load associated with NAROCASS 1 and NAROCASS 2 is evidence of their tolerance to both CBSD and CMD. Therefore, these two cassava clones should be utilized in CBSD and CMD management in Uganda, including their utilization as progenitors in further virus resistance breeding.

Localization of cassava brown streak virus in Nicotiana rustica and cassava Manihot esculenta (Crantz) using RNAscope® in situ hybridization.

BACKGROUND: Cassava brown streak disease (CBSD) has a viral aetiology and is caused by viruses belonging to the genus Ipomovirus (family Potyviridae), Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Molecular and serological methods are available for detection, discrimination and quantification of cassava brown streak viruses (CBSVs) in infected plants. However, precise determination of the viral RNA localization in infected host tissues is still not possible pending appropriate methods. RESULTS: We have developed an in situ hybridization (ISH) assay based on RNAscope® technology that allows the sensitive detection and localization of CBSV RNA in plant tissues. The method was initially developed in the experimental host Nicotiana rustica and was then further adapted to cassava. Highly sensitive and specific detection of CBSV RNA was achieved without background and hybridization signals in sections prepared from non-infected tissues. The tissue tropism of CBSV RNAs appeared different between N. rustica and cassava. CONCLUSIONS: This study provides a robust method for CBSV detection in the experimental host and in cassava. The protocol will be used to study CBSV tropism in various cassava genotypes, as well as CBSVs/cassava interactions in single and mixed infections.

The first transcriptomes from field-collected individual whiteflies ( Bemisia tabaci, Hemiptera: Aleyrodidae): a case study of the endosymbiont composition.

Background:Bemisia tabaci species ( B. tabaci), or whiteflies, are the world's most devastating insect pests. They cause billions of dollars (US) of damage each year, and are leaving farmers in the developing world food insecure. Currently, all publically available transcriptome data for B. tabaci are generated from pooled samples, which can lead to high heterozygosity and skewed representation of the genetic diversity. The ability to extract enough RNA from a single whitefly has remained elusive due to their small size and technological limitations. Methods: In this study, we optimised a single whitefly RNA extraction procedure, and sequenced the transcriptome of four individual adult Sub-Saharan Africa 1 (SSA1) B. tabaci. Transcriptome sequencing resulted in 39-42 million raw reads. De novo assembly of trimmed reads yielded between 65,000-162,000 Contigs across B. tabaci transcriptomes. Results: Bayesian phylogenetic analysis of mitochondrion cytochrome I oxidase (mtCOI) grouped the four whiteflies within the SSA1 clade. BLASTn searches on the four transcriptomes identified five endosymbionts; the primary endosymbiont Portieraaleyrodidarum and four secondary endosymbionts: Arsenophonus, Wolbachia, Rickettsia, and Cardinium spp. that were predominant across all four SSA1 B. tabaci samples with prevalence levels of between 54.1 to 75%. Amino acid alignments of the NusG gene of P. aleyrodidarum for the SSA1 B. tabaci transcriptomes of samples WF2 and WF2b revealed an eleven amino acid residue deletion that was absent in samples WF1 and WF2a. Comparison of the protein structure of the NusG protein from P. aleyrodidarum in SSA1 with known NusG structures showed the deletion resulted in a shorter D loop. Conclusions: The use of field-collected specimens means time and money will be saved in future studies using single whitefly transcriptomes in monitoring vector and viral interactions. Our method is applicable to any small organism where RNA quantity has limited transcriptome studies.

African ancestry of New World, Bemisia tabaci-whitefly species.

Bemisia tabaci whitefly species are some of the world's most devastating agricultural pests and plant-virus disease vectors. Elucidation of the phylogenetic relationships in the group is the basis for understanding their evolution, biogeography, gene-functions and development of novel control technologies. We report here the discovery of five new Sub-Saharan Africa (SSA) B. tabaci putative species, using the partial mitochondrial cytochrome oxidase 1 gene: SSA9, SSA10, SSA11, SSA12 and SSA13. Two of them, SSA10 and SSA11 clustered with the New World species and shared 84.8‒86.5% sequence identities. SSA10 and SSA11 provide new evidence for a close evolutionary link between the Old and New World species. Re-analysis of the evolutionary history of B. tabaci species group indicates that the new African species (SSA10 and SSA11) diverged from the New World clade c. 25 million years ago. The new putative species enable us to: (i) re-evaluate current models of B. tabaci evolution, (ii) recognise increased diversity within this cryptic species group and (iii) re-estimate divergence dates in evolutionary time.

Exchanging and managing in-vitro elite germplasm to combat Cassava Brown Streak Disease (CBSD) and Cassava Mosaic Disease (CMD) in Eastern and Southern Africa.

Cassava varieties resistant to cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are needed for the food and income security of the rural poor in eastern and southern Africa (ESA). The International Institute of Tropical Agriculture led five national cassava breeding programs (Malawi, Mozambique, Kenya, Tanzania and Uganda) in virus-cleaning and exchanging elite cassava germplasm resistant to both diseases. This paper documents the experiences and lessons learned from the process. Thirty-one clones (25 elite, two standard and four national) were submitted by the five breeding programs to the Natural Resources Institute and Kenya Plant Health Inspectorate Services for virus cleaning and indexing. Subsequently, ca 75 invitro virus-indexed plantlets per clone were sent to Genetic Technologies International Limited (GTIL), a private tissue culture (TC) lab in Kenya, and micro-propagated to produce ≥1500 plantlets. After fulfilling all the formal procedures of germplasm exchange between countries ≥300 plantlets per clone were sent to each partner country. National check clones susceptible to CMD/CBSD were sent only to their countries of origin. In each country, the in-vitro plantlets were acclimatized under screen house conditions and transferred to clean isolated sites for field multiplication. All the clones were cleaned of the viruses, except Tomo. The cleaning process was slow for F19-NL, NASE1, and Kibandameno and TC micro-propagation at GTIL was less efficient for Pwani, Tajirika, NASE1, and Okhumelela than for the other clones. Difficulties in cleaning recalcitrant clones affected the timeline for establishing the multi-site evaluation trials in target countries. The initiative is the one of the kind to successfully clean and exchange elite germplasm as a joint action to combat CBSD in ESA. Adequate preparation in terms of infrastructure and personnel are critical to successfully receiving and adapting the indexed in-vitro plants as new germplasm.

Efficacy of chemotherapy and thermotherapy in elimination of East African cassava mosaic virus from Tanzanian cassava landrace.

Cassava mosaic disease is caused by cassava mosaic begomoviruses (CMBs) and can result in crop losses up to 100% in cassava (Manihot esculenta) in Tanzania. We investigated the efficacy of chemotherapy and thermotherapy for elimination of East African cassava mosaic virus (EACMV) of Tanzanian cassava. In vitro plantlets from EACMV-infected plants obtained from coastal Tanzania were established in the greenhouse. Leaves were sampled from the plants and tested to confirm the presence of EACMV. Plantlets of plants positive for EACMV were initiated in Murashige and Skoog (MS) medium. On the second subculture, they were subjected into chemical treatment in the medium containing salicylic acid (0, 10, 20, 30 and 40 mg/L) and ribavirin (0, 5, 10, 15 and 20 mg/L). In the second experiment, EACMV-infected plantlets were subjected to temperatures between 35 and 40°C with 28°C as the control. After 42 days of growth, DNA was extracted from plant leaves and PCR amplification was performed using EACMV specific primers. It was found that plant survival decreased with increasing levels of both salicylic acid and ribavirin concentrations. In general, plants treated with salicylic acid exhibited a lower plant survival % than those treated with ribavirin. However, the percentage of virus-free plants increased with an increase in the concentration of both ribavirin and salicylic acid. The most effective concentrations were 20 mg/L of ribavirin and 30 mg/L of salicylic acid; these resulted in 85.0% and 88.9% virus-free plantlets, respectively. With regard to thermotherapy, 35°C resulted in 79.5% virus-free plantlets compared to 69.5% at 40°C. Based on virus elimination, ribavirin at 20 mg/L, salicylic acid 30 mg/L and thermotherapy at 35°C are recommended for production of EACMV free cassava plantlets from infected cassava landraces.

Unusual occurrence of a DAG motif in the Ipomovirus Cassava brown streak virus and implications for its vector transmission.

Cassava is the main staple food for over 800 million people globally. Its production in eastern Africa is being constrained by two devastating Ipomoviruses that cause cassava brown streak disease (CBSD); Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), with up to 100% yield loss for smallholder farmers in the region. To date, vector studies have not resulted in reproducible and highly efficient transmission of CBSV and UCBSV. Most virus transmission studies have used Bemisia tabaci (whitefly), but a maximum of 41% U/CBSV transmission efficiency has been documented for this vector. With the advent of next generation sequencing, researchers are generating whole genome sequences for both CBSV and UCBSV from throughout eastern Africa. Our initial goal for this study was to characterize U/CBSV whole genomes from CBSD symptomatic cassava plants sampled in Kenya. We have generated 8 new whole genomes (3 CBSV and 5 UCBSV) from Kenya, and in the process of analyzing these genomes together with 26 previously published sequences, we uncovered the aphid transmission associated DAG motif within coat protein genes of all CBSV whole genomes at amino acid positions 52-54, but not in UCBSV. Upon further investigation, the DAG motif was also found at the same positions in two other Ipomoviruses: Squash vein yellowing virus (SqVYV), Coccinia mottle virus (CocMoV). Until this study, the highly-conserved DAG motif, which is associated with aphid transmission was only noticed once, in SqVYV but discounted as being of minimal importance. This study represents the first comprehensive look at Ipomovirus genomes to determine the extent of DAG motif presence and significance for vector relations. The presence of this motif suggests that aphids could potentially be a vector of CBSV, SqVYV and CocMov. Further transmission and ipomoviral protein evolutionary studies are needed to confirm this hypothesis.

A VIGS screen identifies immunity in the Arabidopsis Pla-1 accession to viruses in two different genera of the Geminiviridae.

Geminiviruses are DNA viruses that cause severe crop losses in different parts of the world, and there is a need for genetic sources of resistance to help combat them. Arabidopsis has been used as a source for virus-resistant genes that derive from alterations in essential host factors. We used a virus-induced gene silencing (VIGS) vector derived from the geminivirus Cabbage leaf curl virus (CaLCuV) to assess natural variation in virus-host interactions in 190 Arabidopsis accessions. Silencing of CH-42, encoding a protein needed to make chlorophyll, was used as a visible marker to discriminate asymptomatic accessions from those showing resistance. There was a wide range in symptom severity and extent of silencing in different accessions, but two correlations could be made. Lines with severe symptoms uniformly lacked extensive VIGS, and lines that showed attenuated symptoms over time (recovery) showed a concomitant increase in the extent of VIGS. One accession, Pla-1, lacked both symptoms and silencing, and was immune to wild-type infectious clones corresponding to CaLCuV or Beet curly top virus (BCTV), which are classified in different genera in the Geminiviridae. It also showed resistance to the agronomically important Tomato yellow leaf curl virus (TYLCV). Quantitative trait locus mapping of a Pla-1 X Col-0 F2 population was used to detect a major peak on chromosome 1, which is designated gip-1 (geminivirus immunity Pla-1-1). The recessive nature of resistance to CaLCuV and the lack of obvious candidate genes near the gip-1 locus suggest that a novel resistance gene(s) confers immunity.