Efficient CRISPR/Cas9 genome editing of Phytoene desaturase in cassava

CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (Manihot esculenta), the Phytoene desaturase (MePDS) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium-mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted MePDS site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 5 0 and or downstream of the 3 0 targeted MePDS region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of MePDS described here generates visually detectable mutated events in a relatively short time frame of 6-8 weeks, and does not require sequencing to confirm editing at the target. It therefore provides a valuable platform to facilitate rapid assessment and optimization of CRISPR/Cas9 and other genome-editing technologies in cassava

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

In silico prediction of candidate gene targets for the management of African cassava whitefly (Bemisia tabaci, SSA1-SG1), a key vector of viruses causing cassava brown streak disease

Whiteflies (Bemisia tabaci sensu lato) have a wide host range and are globally important agricultural pests. In Sub-Saharan Africa, they vector viruses that cause two ongoing disease epidemics: cassava brown streak disease and cassava mosaic virus disease. These two diseases threaten food security for more than 800 million people in Sub-Saharan Africa. Efforts are ongoing to identify target genes for the development of novel management options against the whitefly populations that vector these devastating viral diseases affecting cassava production in Sub-Saharan Africa. This study aimed to identify genes that mediate osmoregulation and symbiosis functions within cassava whitefly gut and bacteriocytes and evaluate their potential as key gene targets for novel whitefly control strategies. The gene expression profiles of dissected guts, bacteriocytes and whole bodies were compared by RNAseq analysis to identify genes with significantly enriched expression in the gut and bacteriocytes. Phylogenetic analyses identified three candidate osmoregulation gene targets: two α-glucosidases, SUC 1 and SUC 2 with predicted function in sugar transformations that reduce osmotic pressure in the gut; and a water-specific aquaporin (AQP1) mediating water cycling from the distal to the proximal end of the gut. Expression of the genes in the gut was enriched 23.67-, 26.54- and 22.30-fold, respectively. Genome-wide metabolic reconstruction coupled with constraint-based modeling revealed four genes (argH, lysA, BCAT & dapB) within the bacteriocytes as potential targets for the management of cassava whiteflies. These genes were selected based on their role and essentiality within the different essential amino acid biosynthesis pathways. A demonstration of candidate osmoregulation and symbiosis gene targets in other species of the Bemisia tabaci species complex that are orthologs of the empirically validated osmoregulation genes highlights the latter as promising gene targets for the control of cassava whitefly pests by in planta RNA interference

Detection of cassava brown streak ipomoviruses in aphids collected from cassava plants

Cassava is an important staple food in Africa and a major source of carbohydrates for 800 million people globally. However, cassava suffers severe yield losses caused by many factors including pests and diseases. A devastating disease of cassava is cassava brown streak disease (CBSD) caused by the cassava brown streak ipomoviruses (CBSIs) (family Potyviridae), Cassava brown streak virus (CBSV), and Ugandan cassava brown streak virus (UCBSV). Spread of CBSD is mainly through planting infected stem cuttings used for propagation. Transmission of CBSIs by the insect vector (Bemisia tabaci) has been reported. However, experimental transmission efficiencies of CBSIs are usually low. Recent research has showed the occurrence of a DAG motif associated with aphid transmission in other potyviruses, within the coat protein gene of CBSV. Consequently this study aimed to explore the possibility that besides whiteflies, aphids may transmit CBSIs. Cassava plants were assessed during a survey for occurrence of CBSD and aphids as potential alternative CBSIs vectors. We collected aphids from CBSD-symptomatic and symptomless cassava plants within farmers' fields in Uganda during April–July 2020. The aphids were analyzed for the presence of CBSIs by reverse transcriptase-polymerase chain reaction (RT-PCR) and to determine aphid species using mitochondrial cytochrome oxidase (mtCOI) barcoding. Unusual aphid infestation of cassava plants was observed at 35 locations in nine districts across Uganda and on 11 other plant species within or adjacent to cassava fields. This is the first report of aphids infesting cassava in Uganda. Molecular analysis of the aphid confirmed presence of three different aphid species in the surveyed cassava fields, namely, Aphis solanella, Aphis fabae mordvilkoi, and Rhopalosiphum sp. mtCOI nucleotide sequences for the aphids in which CBSIs were detected are deposited with Genbank under accession numbers OP223337-40. Both UCBSV and CBSV were detected by RT-PCR in aphids collected from cassava fields with CBSD-affected plants. The CBSIs were detected in 14 aphid samples collected from 19 CBSD-symptomatic cassava plants. These results suggest the ability of aphids to acquire CBSIs, but transmission experiments are required on their vector potential

Occurrence of Bondar's Nesting Whitefly, Paraleyrodes bondari (Hemiptera: Aleyrodidae), on cassava in Uganda

Cassava is a valued calorific source to millions of Africans who eat it daily and a vital staple for their foodsecurity. One of the key constraints to this crop is whiteflies which are both a vector of viral diseasesand a direct pest. Although the African cassava whitefly is known to cause physical damage on cassavawith considerable tuberous yield loss, a recent whitefly outbreak caused unusually severe damage, whichprompted the current reported investigation. Molecular identification of whitefly adults sampled fromthe affected cassava field revealed the presence of a new whitefly species, Paraleyrodes bondari. Thiscommunication is the first report of the occurrence of P. bondari on cassava in Uganda

Genetic diversity of whitefly (Bemisia spp.) on crop and uncultivated plants in Uganda: implications for the control of this devastating pest species complex in Africa

Over the past three decades, highly increased whitefly (Bemisia tabaci) populations have been observed on the staple food crop cassava in eastern Africa and associated with ensuing viral disease pandemics and food insecurity. Increased whitefly numbers have also been observed in other key agricultural crops and weeds. Factors behind the population surges on different crops and their interrelationships are unclear, although in cassava they have been associated with specific populations within the Bemisia tabaci species complex known to infest cassava crops in Africa. This study carried out an in-depth survey to understand the distribution of B. tabaci populations infesting crops and uncultivated plant hosts in Uganda, a centre of origin for this pest complex. Whitefly samples were collected from 59 identified plant species and 25 unidentified weeds in a countrywide survey. Identities of 870 individual adult whiteflies were determined through mitochondrial cytochrome oxidase 1 sequences (651 bp) in the 3′ barcode region used for B. tabaci systematics. Sixteen B. tabaci and five related whitefly putative species were identified based on > 4.0% nucleotide divergence, of which three are proposed as novel B. tabaci putative species and four as novel closely related whitefly species. The most prevalent whiteflies were classified as B. tabaci MED-ASL (30.5% of samples), sub-Saharan Africa 1 (SSA1, 22.7%) and Bemisia Uganda1 (12.1%). These species were also indicated to be the most polyphagous occurring on 33, 40 and 25 identified plant species, respectively. Multiple (≥ 3) whitefly species occurred on specific crops (bean, eggplant, pumpkin and tomato) and weeds (Sida acuta and Ocimum gratissimum). These plants may have increased potential to act as reservoirs for mixed infections of whitefly-vectored viruses. Management of whitefly pest populations in eastern Africa will require an integration of approaches that consider their degree of polyphagy and a climate that enables the continuous presence of crop and uncultivated plant hosts

Review and guide to a future naming system of African Bemisia tabaci species

Once a pest has been correctly identified, its genus and species name can provide a link to valuable indications of its ecology, biology and life history that are critical for developing control strategies. Importantly, this link should exist even when the pest was known under other names (synonyms), or was not considered a pest at all (National Research Council, 1968). Many examples have shown that incorrect identification or classification of a pest has led to fruitless searches for biocontrol agents in the native range, incorrect assignments as disease vectors, and costly, yet misdirected, suppression measures. As new approaches for delimiting species based on molecular information become more widely used, the process of correctly identifying a species has become even more complex. Fortunately, we have good systematic frameworks and nomenclatural systems that are able to cope with these challenges. Here we review challenges associated with classification and identification within the Bemisia tabaci (Gennadius) species complex. These pests and the viruses they transmit have emerged in the past few decades as among the most damaging to food and fibre crops globally (Varma & Malathi, 2003; Pimental et al., 2005; Seal et al., 2006), especially in sub‐Saharan Africa (SSA). The systematics of the B. tabaci species group has been a highly debated topic for years (Boykin, 2014). Putative species are indistinguishable morphologically, so other biological data have been collected to investigate the species in the complex. Based on genetic differences (Colvin et al., 2004; Sseruwagi et al., 2005; Boykin et al., 2007; Boykin et al., 2013; Hsieh et al., 2014) and mating incompatibility (Colvin et al., 2004; Liu et al., 2007; Xu et al., 2010), B. tabaci is now recognized as a species complex that consists of at least 34 putative species (Boykin et al., 2012). The rapid discovery of significant species diversity has led to many changes in the informal names used over the last 10 years (Boykin, 2014), creating confusion in the literature

A novel seed treatment-based multiplication approach for cassava planting material.

Cassava (Manihot esculenta Crantz) is an important food security crop in many parts of the developing world. The crop?s high yield potential and multitude of uses?both for nutrition and processing?render cassava a promising driver for the development of rural value chains. It is traditionally propagated from stem cuttings of up to 30 cm in length, giving a multiplication rate as low as 1:10. Propagating cassava traditionally is very inefficient, which leads to challenges in the production and distribution of quality planting material and improved cultivars, greatly limiting the impact of investments in crop breeding. The work described in the present study aimed to develop a seed treatment approach to facilitate the use of shorter seed pieces, increasing the multiplication rate of cassava and thus making the crop?s seed systems more efficient. After several tests, formulation was identified, consisting of thiamethoxam 21 g ha-1, mefenoxam 1.0 g ha-1, fludioxonil 1.3 g ha-1, thiabendazole 7.5 g ha-1 and Latex 2% as a binder. Plant growing from seed pieces treated with this formulation displayed increased crop establishment and early crop vigor, leading to an improved productivity throughout a full growing cycle. This allowed to reduce the cassava seed piece size to 8 cm with no negative effects on germination and crop establishment, leading to yields comparable to those from untreated 16 cm pieces. This, in turn, will allow to increase the multiplication ratio of cassava by a factor of up to 3. Notably, this was possible under regular field conditions and independently of any specialised treatment facilities. Compared with existing seed production protocols, the increased multiplication rates allowed for efficiency gains of between 1 to 1.9 years compared to conventional five-year cycles. We believe that the technology described here holds considerable promise for developing more reliable and remunerative delivery channels for quality cassava planting material and improved genetics