Genome-wide analyses of cassava Pathogenesis-related (PR) gene families reveal core transcriptome responses to whitefly infestation, salicylic acid and jasmonic acid.

BACKGROUND: Whiteflies are a threat to cassava (Manihot esculenta), an important staple food in many tropical/subtropical regions. Understanding the molecular mechanisms regulating cassava's responses against this pest is crucial for developing control strategies. Pathogenesis-related (PR) protein families are an integral part of plant immunity. With the availability of whole genome sequences, the annotation and expression programs of the full complement of PR genes in an organism can now be achieved. An understanding of the responses of the entire complement of PR genes during biotic stress and to the defense hormones, salicylic acid (SA) and jasmonic acid (JA), is lacking. Here, we analyze the responses of cassava PR genes to whiteflies, SA, JA, and other biotic aggressors. RESULTS: The cassava genome possesses 14 of the 17 plant PR families, with a total of 447 PR genes. A cassava PR gene nomenclature is proposed. Phylogenetic relatedness of cassava PR proteins to each other and to homologs in poplar, rice and Arabidopsis identified cassava-specific PR gene family expansions. The temporal programs of PR gene expression in response to the whitefly (Aleurotrachelus socialis) in four whitefly-susceptible cassava genotypes showed that 167 of the 447 PR genes were regulated after whitefly infestation. While the timing of PR gene expression varied, over 37% of whitefly-regulated PR genes were downregulated in all four genotypes. Notably, whitefly-responsive PR genes were largely coordinately regulated by SA and JA. The analysis of cassava PR gene expression in response to five other biotic stresses revealed a strong positive correlation between whitefly and Xanthomonas axonopodis and Cassava Brown Streak Virus responses and negative correlations between whitefly and Cassava Mosaic Virus responses. Finally, certain associations between PR genes in cassava expansions and response to biotic stresses were observed among PR families. CONCLUSIONS: This study represents the first genome-wide characterization of PR genes in cassava. PR gene responses to six biotic stresses and to SA and JA are demonstrably different to other angiosperms. We propose that our approach could be applied in other species to fully understand PR gene regulation by pathogens, pests and the canonical defense hormones SA and JA.

A metabolomics characterisation of natural variation in the resistance of cassava to whitefly.

BACKGROUND: Cassava whitefly outbreaks were initially reported in East and Central Africa cassava (Manihot esculenta Crantz) growing regions in the 1990's and have now spread to other geographical locations, becoming a global pest severely affecting farmers and smallholder income. Whiteflies impact plant yield via feeding and vectoring cassava mosaic and brown streak viruses, making roots unsuitable for food or trading. Deployment of virus resistant varieties has had little impact on whitefly populations and therefore development of whitefly resistant varieties is also necessary as part of integrated pest management strategies. Suitable sources of whitefly resistance exist in germplasm collections that require further characterization to facilitate and assist breeding programs. RESULTS: In the present work, a hierarchical metabolomics approach has been employed to investigate the underlying biochemical mechanisms associated with whitefly resistance by comparing two naturally occurring accessions of cassava, one susceptible and one resistant to whitefly. Quantitative differences between genotypes detected at pre-infestation stages were consistently observed at each time point throughout the course of the whitefly infestation. This prevalent differential feature suggests that inherent genotypic differences override the response induced by the presence of whitefly and that they are directly linked with the phenotype observed. The most significant quantitative changes relating to whitefly susceptibility were linked to the phenylpropanoid super-pathway and its linked sub-pathways: monolignol, flavonoid and lignan biosynthesis. These findings suggest that the lignification process in the susceptible variety is less active, as the susceptible accession deposits less lignin and accumulates monolignol intermediates and derivatives thereof, differences that are maintained during the time-course of the infestation. CONCLUSIONS: Resistance mechanism associated to the cassava whitefly-resistant accession ECU72 is an antixenosis strategy based on reinforcement of cell walls. Both resistant and susceptible accessions respond differently to whitefly attack at biochemical level, but the inherent metabolic differences are directly linked to the resistance phenotype rather than an induced response in the plant.

Next-generation sequencing reveals endosymbiont variability in cassava whitefly, Bemisia tabaci, across the agro-ecological zones of Kerala, India.

Silverleaf whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is one of the most notorious invasive insect pests, infesting more than 900 species of plants and spreading more than 200 viral diseases. This polyphagous agricultural pest harbours diverse bacterial communities in its gut, which perform multiple functions in whiteflies, including nutrient provisioning, amino acid biosynthesis, and virus transmission. The present exploratory study compares the bacterial communities associated with silverleaf whitefly infesting cassava, also known as cassava whitefly, collected from two different zones (zone P: plains; zone H: high ranges), from Kerala, India, using next-generation sequencing of 16S rDNA. The data sets for these two regions consisted of 1 321 906 and 690 661 high-quality paired-end sequences with mean length of 150 bp. Highly diverse bacterial communities were present in the sample, containing approximately 3513 operational taxonomic units (OTUs). Sequence analysis showed a marked difference in the relative abundance of bacteria in the populations. A total of 16 bacterial phyla, 27 classes, 56 orders, 91 families, 236 genera, and 409 species were identified from the P population, against 16, 31, 60, 88, 225, and 355, respectively, in the H population. Arsenophonus sp. (Enterobacteriaceae), which is important for virus transmission by whiteflies, was relatively abundant in the P population, whereas in the H population Bacillus sp. was the most dominant group. The association of whitefly biotypes and secondary symbionts suggests a possible contribution of these bacteria to host characteristics such as virus transmission, host range, insecticide resistance, and speciation.

Differential expression of the Hsf family in cassava under biotic and abiotic stresses.

Heat shock transcription factors (Hsfs) are important regulators of biotic and abiotic stress responses in plants. Currently, the Hsf gene family is not well understood in cassava, an important tropical crop. In the present study, 32 MeHsf genes were identified from the cassava genome database, which were divided into three types based on functional domain and motif distribution analyses. Analysis of the differential expression of the genes belonging to the Hsf family in cassava was carried out based on published cassava transcriptome data from tissues/organs (leaf blade, leaf midvein, lateral buds, organized embryogenic structures, friable embryogenic callus, fibrous roots, storage roots, stem, petiole, shoot apical meristem, and root apical meristem) under abiotic stress (cold, drought) or biotic stress (mealybugs. cassava brown streak disease, cassava bacterial blight). The results show the expression diversity of cassava Hsfs genes in various tissues/organs. The transcript levels of MeHsfB3a, MeHsfA6a, MeHsfA2a, and MeHsfA9b were upregulated by abiotic and biotic stresses, such as cold, drought, cassava bacterial blight, cassava brown streak disease, and mealybugs, indicating their potential roles in mediating the response of cassava plants to environment stresses. Further interaction network and co-expression analyses suggests that Hsf genes may interact with Hsp70 family members to resist environmental stresses in cassava. These results provide valuable information for future studies of the functional characterization of the MeHsf gene family.

Increased activities of peroxidase and polyphenol oxidase enhance cassava resistance to Tetranychus urticae.

In order to study the function of peroxidase (POD) and polyphenol oxidase (PPO) in cassava resistance to spider mites, we tested the changes of transcription levels and activities of these two protective enzymes in both cassava and Tetranychus urticae (=T. cinnabarinus) during the interaction. The results showed that after damage of the mite-susceptible cassava cultivar BRA900 by T. urticae for 1 and 8 days, the transcription levels of MePOD and MePPO and the activities of POD and PPO showed no significant difference compared with those in undamaged leaves. However, the corresponding transcription levels and activities in 1- and 8-day-damaged leaves of mite-resistant cassava cultivar C1115 increased to a significant level of approximately twofold. When T. urticae fed on BRA900 for 1 and 8 days, the transcription levels of TcPPO and TcPOD and the activities of PPO and POD showed no significant difference compared with those before feeding. However, the corresponding transcription levels and activities of these two protective enzymes in T. urticae feeding on C1115 significantly decreased by about half. This study preliminarily validates the function of POD and PPO in cassava resistance to T. urticae, and provides candidate gene resource for molecular breeding of spider mite-resistant cassava.

Prevalence and genetic diversity of endosymbiotic bacteria infecting cassava whiteflies in Africa.

BACKGROUND: Cassava provides over half of the dietary requirement for more than 200 million poor in Africa. In recent years, cassava has been affected by an epidemic of a virus disease called cassava brown streak disease (CBSD) that is spreading in much of eastern and central Africa, affecting food security and the economic development of the poor. The viruses that cause CBSD are transmitted by the insect vector whitefly (Bemisia tabaci), which have increased to very high numbers in some African countries. Strains of endosymbiotic bacteria infecting whiteflies have been reported to interact specifically with different whitefly populations with varied effects on its host biology and efficiency of virus transmission. The main aim of this study was therefore to investigate the prevalence and diversity of the secondary endosymbiotic bacteria infecting cassava whiteflies with a view to better understand their role on insect population dynamics and virus disease epidemics. RESULTS: The genetic diversity of field-collected whitefly from Tanzania, Malawi, Uganda and Nigeria was determined by mitochondrial DNA based phylogeny and restriction fragment length polymorphism. Cassava in these countries was infected with five whitefly populations, and each one was infected with different endosymbiotic bacteria. Incidences of Arsenophonus, Rickettsia, Wolbachia and Cardinium varied amongst the populations. Wolbachia was the most predominant symbiont with infection levels varying from 21 to 97%. Infection levels of Arsenophonus varied from 17 to 64% and that of Rickettsia was 0 to 53%. Hamiltonella and Fritschea were absent in all the samples. Multiple locus sequence typing identified four different strains of Wolbachia infecting cassava whiteflies. A common strain of Wolbachia infected the whitefly population Sub-Saharan Africa 1-subgroup 1 (SSA1-SG1) and SSA1-SG2, while others were infected with different strains. Phylogeny based on 16S rDNA of Rickettsia and 23S rDNA of Arsenophonus also identified distinct strains. CONCLUSIONS: Genetically diverse bacteria infect cassava whiteflies in Africa with varied prevalence across different host populations, which may affect their whitefly biology. Further studies are required to investigate the role of endosymbionts to better understand the whitefly population dynamics.

The Dynamics and Environmental Influence on Interactions Between Cassava Brown Streak Disease and the Whitefly, Bemisia tabaci.

Cassava brown streak disease (CBSD) is currently the most significant virus disease phenomenon affecting African agriculture. In this study, we report results from the most extensive set of field data so far presented for CBSD in Africa. From assessments of 515 farmers' plantings of cassava, incidence in the Coastal Zone of Tanzania (46.5% of plants; 87% of fields affected) was higher than in the Lake Zone (22%; 34%), but incidences for both zones were greater than previous published records. The whitefly vector, Bemisia tabaci, was more abundant in the Lake Zone than the Coastal Zone, the reverse of the situation reported previously, and increased B. tabaci abundance is driving CBSD spread in the Lake Zone. The altitudinal "ceiling" previously thought to restrict the occurrence of CBSD to regions <1,000 masl has been broken as a consequence of the greatly increased abundance of B. tabaci in mid-altitude areas. Among environmental variables analyzed, minimum temperature was the strongest determinant of CBSD incidence. B. tabaci in the Coastal and Lake Zones responded differently to environmental variables examined, highlighting the biological differences between B. tabaci genotypes occurring in these regions and the superior adaptation of B. tabaci in the Great Lakes region both to cassava and low temperature conditions. Regression analyses using multi-country data sets could be used to determine the potential environmental limits of CBSD. Approaches such as this offer potential for use in the development of predictive models for CBSD, which could strengthen country- and continent-level CBSD pandemic mitigation strategies.

Genome sequence of Erinnyis ello granulovirus (ErelGV), a natural cassava hornworm pesticide and the first sequenced sphingid-infecting betabaculovirus.

BACKGROUND: Cassava (Manihot esculenta) is the basic source for dietary energy of 500 million people in the world. In Brazil, Erinnyis ello ello (Lepidoptera: Sphingidae) is a major pest of cassava crops and a bottleneck for its production. In the 1980s, a naturally occurring baculovirus was isolated from E. ello larva and successfully applied as a bio-pesticide in the field. Here, we described the structure, the complete genome sequence, and the phylogenetic relationships of the first sphingid-infecting betabaculovirus. RESULTS: The baculovirus isolated from the cassava hornworm cadavers is a betabaculovirus designated Erinnyis ello granulovirus (ErelGV). The 102,759 bp long genome has a G + C content of 38.7%. We found 130 putative ORFs coding for polypeptides of at least 50 amino acid residues. Only eight genes were found to be unique. ErelGV is closely related to ChocGV and PiraGV isolates. We did not find typical homologous regions and cathepsin and chitinase homologous genes are lacked. The presence of he65 and p43 homologous genes suggests horizontal gene transfer from Alphabaculovirus. Moreover, we found a nucleotide metabolism-related gene and two genes that could be acquired probably from Densovirus. CONCLUSIONS: The ErelGV represents a new virus species from the genus Betabaculovirus and is the closest relative of ChocGV. It contains a dUTPase-like, a he65-like, p43-like genes, which are also found in several other alpha- and betabaculovirus genomes, and two Densovirus-related genes. Importantly, recombination events between insect viruses from unrelated families and genera might drive baculovirus genomic evolution.

Current and future scenarios of suitability and expansion of cassava brown streak disease, Bemisia tabaci species complex, and cassava planting in Africa.

Cassava (Manihot esculenta) is among the most important staple crops globally, with an imperative role in supporting the Sustainable Development Goal of 'Zero hunger'. In sub-Saharan Africa, it is cultivated mainly by millions of subsistence farmers who depend directly on it for their socio-economic welfare. However, its yield in some regions has been threatened by several diseases, especially the cassava brown streak disease (CBSD). Changes in climatic conditions enhance the risk of the disease spreading to other planting regions. Here, we characterise the current and future distribution of cassava, CBSD and whitefly Bemisia tabaci species complex in Africa, using an ensemble of four species distribution models (SDMs): boosted regression trees, maximum entropy, generalised additive model, and multivariate adaptive regression splines, together with 28 environmental covariates. We collected 1,422 and 1,169 occurrence records for cassava and Bemisia tabaci species complex from the Global Biodiversity Information Facility and 750 CBSD occurrence records from published literature and systematic surveys in East Africa. Our results identified isothermality as having the highest contribution to the current distribution of cassava, while elevation was the top predictor of the current distribution of Bemisia tabaci species complex. Cassava harvested area and precipitation of the driest month contributed the most to explain the current distribution of CBSD outbreaks. The geographic distributions of these target species are also expected to shift under climate projection scenarios for two mid-century periods (2041-2060 and 2061-2080). Our results indicate that major cassava producers, like Cameron, Ivory Coast, Ghana, and Nigeria, are at greater risk of invasion of CBSD. These results highlight the need for firmer agricultural management and climate-change mitigation actions in Africa to combat new outbreaks and to contain the spread of CBSD.

Occurrence of lace bug Vatiga illudens and Vatiga manihotae (Hemiptera: Tingidae) in Mato Grosso do Sul, midwestern Brazil.

Nymphs and adults of the lace bug (Hemiptera: Tingidae) have been found in cassava crops (Manihot esculenta) in Mato Grosso do Sul State, Brazil. The insects were collected in the field and taken to the laboratory where they were identified based on some morphological traits of the species Vatiga manihotae (Drake) and V. illudens (Drake), which are first reported in the aforementioned state.

Molecular detection of establishment and geographical distribution of Brazilian isolates of Neozygites tanajoae, a fungus pathogenic to cassava green mite, in Benin (West Africa).

Diagnostic PCR with two specific primer pairs (NEOSSU and 8DDC) were used to monitor the establishment and geographical distribution of Brazilian isolates of Neozygites tanajoae Delalibera, Hajek and Humber (Entomophthorales: Neozygitaceae) released in Benin for the biological control of the cassava green mite, Mononychellus tanajoa (Bondar) (Acari: Tetranychidae). A total of 141 cassava fields were visited and samples of M. tanajoa suspected to be infected by N. tanajoae were collected in 60 fields distributed between the coastal Southern Forest Mosaic (SFM) and the Northern Guinea Savanna (NGS) zones of Benin, West Africa. Analysis of DNA samples of dead mites using the species specific NEOSSU primers revealed the presence of N. tanajoae in 46 fields. The second country specific pair of primers 8DDC revealed the presence of Brazilian isolates of N. tanajoae in 36 fields, representing 78.3% of fields positive for N. tanajoae. Brazilian isolates occurred from SFM to NGS zones in Benin, however, they were concentrated in fields located within former release zones (e.g. Department of Ouémé in the South and Borgou in the North). In contrast, the indigenous African isolates of N. tanajoae were evenly distributed in the sub-humid and humid savannah zones of the country. The mean infection rate of M. tanajoa with indigenous isolates of N. tanajoae was relatively low (5.3%) compared to Brazilian isolates (28%), indicating a higher biocontrol potential of the latter. This first post-release monitoring using PCR techniques showed that the Brazilian strains of N. tanajoae is well established in Benin and spread effectively in this area.

On species delimitation, hybridization and population structure of cassava whitefly in Africa.

The Bemisia cassava whitefly complex includes species that cause severe crop damage through vectoring cassava viruses in eastern Africa. Currently, this whitefly complex is divided into species and subgroups (SG) based on very limited molecular markers that do not allow clear definition of species and population structure. Based on 14,358 genome-wide SNPs from 62 Bemisia cassava whitefly individuals belonging to sub-Saharan African species (SSA1, SSA2 and SSA4), and using a well-curated mtCOI gene database, we show clear incongruities in previous taxonomic approaches underpinned by effects from pseudogenes. We show that the SSA4 species is nested within SSA2, and that populations of the SSA1 species comprise well-defined south-eastern (Madagascar, Tanzania) and north-western (Nigeria, Democratic Republic of Congo, Burundi) putative sub-species. Signatures of allopatric incipient speciation, and the presence of a 'hybrid zone' separating the two putative sub-species were also detected. These findings provide insights into the evolution and molecular ecology of a highly cryptic hemipteran insect complex in African, and allow the systematic use of genomic data to be incorporated in the development of management strategies for this cassava pest.

Resistance to the whitefly, Aleurotrachelus socialis, in wild populations of cassava, Manihot tristis.

The levels of resistance in the wild species of cassava, Manihot tristis Muell-Arg. (Malpighiales: Euphorbiaceae), to the whitefly, Aleurotrachelus socialis Bondar (Hemiptera: Alelyrodidae), the most important economic pest in cassava, Manihot esculenta Crantz (Malpighiales: Euphorbiaceae) crops in South America, were estimated under glasshouse conditions. The parameters of the life history of A. socialis were studied on TST-26 and TST-18 accessions of the wild parent and compared with the susceptible (CMC-40) and resistant (MEcu-72) cultivars. The average longevity on the wild accessions (TST-26, 4.1; TST-18, 4.6 days) and oviposition rates (TST-26, 2.0; TST-18, 1.6 eggs/female/2 days) of the A. socialis females were not significantly different from those of MEcu-72 (5.1 days and 3.4 eggs/female/2 days). The longevity and oviposition rates on CMC-40 were highest (11 days and 8.6 eggs/female/2 days). Analyses of the demographic parameters (Ro, r(m); DT) showed a significant impact of the M. tristis accessions on the potential growth of A. socialis. The average survival time of adults that fed on TST-26, TST-18, and MEcu-72 were significantly different from those recorded on the susceptible genotype. Results from this study revealed important levels of resistance to the whitefly A. socialis on the TST-26 and TST-18 accessions due to the marked differences found for longevity and reproduction, which influenced and were consistent with the differences found in the net reproduction rate (Ro), intrinsic growth rate (r(m)) and population doubling time (DT). The combined effect of these parameters indicated that M. tristis accessions were inappropriate hosts for A. socialis.

Improving climate suitability for Bemisia tabaci in East Africa is correlated with increased prevalence of whiteflies and cassava diseases.

Projected climate changes are thought to promote emerging infectious diseases, though to date, evidence linking climate changes and such diseases in plants has not been available. Cassava is perhaps the most important crop in Africa for smallholder farmers. Since the late 1990's there have been reports from East and Central Africa of pandemics of begomoviruses in cassava linked to high abundances of whitefly species within the Bemisia tabaci complex. We used CLIMEX, a process-oriented climatic niche model, to explore if this pandemic was linked to recent historical climatic changes. The climatic niche model was corroborated with independent observed field abundance of B. tabaci in Uganda over a 13-year time-series, and with the probability of occurrence of B. tabaci over 2 years across the African study area. Throughout a 39-year climate time-series spanning the period during which the pandemics emerged, the modelled climatic conditions for B. tabaci improved significantly in the areas where the pandemics had been reported and were constant or decreased elsewhere. This is the first reported case where observed historical climate changes have been attributed to the increase in abundance of an insect pest, contributing to a crop disease pandemic.

A simple PCR-based method for the rapid and accurate identification of spider mites (Tetranychidae) on cassava.

The morphological identification of mites entails great challenges. Characteristics such as dorsal setae and aedeagus are widely used, but they show variations between populations, and the technique is time consuming and demands specialized taxonomic expertise that is difficult to access. A successful alternative has been to exploit a region of the mitochondrial cytochrome oxidase I (COI) gene to classify specimens to the species level. We analyzed the COI sequences of four mite species associated with cassava and classified them definitively by detailed morphological examinations. We then developed an identification kit based on the restriction fragment length polymorphism-polymerase chain reaction of subunit I of the COI gene focused on the three restriction enzymes AseI, MboII, and ApoI. This set of enzymes permitted the simple, accurate identification of Mononychellus caribbeanae, M. tanajoa, M. mcgregori, and Tetranychus urticae, rapidly and with few resources. This kit could be a vital tool for the surveillance and monitoring of mite pests in cassava crop protection programs in Africa, Asia, and Latin America.

Identification of additional /novel QTL associated with resistance to cassava green mite in a biparental mapping population.

Cassava green mite [CGM, Mononychellus tanajoa (Bondar)] is the most destructive dry-season pest in most cassava production areas. The pest is responsible for cassava fresh root yield losses of over 80%. Deployment of CGM resistant cultivars is the most cost-effective and sustainable approach of alleviating such production losses. The purposes of this study were to validate the stability of CGM resistance genes found in previously published results, to identify new genes for CGM resistance in bi-parental mapping population and estimate the heritability of the trait. A total of 109 F1 progeny derived from a cross between CGM resistant parent, TMEB778 and a very susceptible parent, TMEB419 were evaluated under CGM hotspot areas in Nigeria for two cropping seasons. A total of 42,204 SNP markers with MAF ≥ 0.05 were used for single-marker analysis. The most significant QTL (S12_7962234) was identified on the left arm on chromosome 12 which explained high phenotypic variance and harboured significant single nucleotide polymorphism (SNP) markers conferring resistance to CGM and leaf pubescence (LP). Colocalization of the most significant SNP associated with resistance to CGM and LP on chromosome 12 is possibly an indication of a beneficial pleiotropic effect or are physically linked. These significant SNPs markers were intersected with the gene annotations and 33 unique genes were identified within SNPs at 4 - 8MB on chromosome 12. Among these genes, nine novel candidate genes namely; Manes.12077600, Manes.12G086200, Manes.12G061200, Manes.12G083100, Manes.12G082000, Manes.12G094100, Manes.12G075600, Manes.12G091400 and Manes.12G069300 highly expressed direct link to cassava green mite resistance. Pyramiding the new QTL/genes identified on chromosome 12 in this study with previously discovered loci, such on chromosome 8, will facilitate breeding varieties that are highly resistant CGM.

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.

Optimal control of a vectored plant disease model for a crop with continuous replanting.

Vector-transmitted diseases of plants have had devastating effects on agricultural production worldwide, resulting in drastic reductions in yield for crops such as cotton, soybean, tomato, and cassava. Plant-vector-virus models with continuous replanting are investigated in terms of the effects of selection of cuttings, roguing, and insecticide use on disease prevalence in plants. Previous models are extended to include two replanting strategies: frequencyreplanting and abundance-replanting. In frequency-replanting, replanting of infected cuttings depends on the selection frequency parameter ε, whereas in abundance-replanting, replanting depends on plant abundance via a selection rate parameter also denoted as ε. The two models are analysed and new thresholds for disease elimination are defined for each model. Parameter values for cassava, whiteflies, and African cassava mosaic virus serve as a case study. A numerical sensitivity analysis illustrates how the equilibrium densities of healthy and infected plants vary with parameter values. Optimal control theory is used to investigate the effects of roguing and insecticide use with a goal of maximizing the healthy plants that are harvested. Differences in the control strategies in the two models are seen for large values of ε. Also, the combined strategy of roguing and insecticide use performs better than a single control.

What has changed in the outbreaking populations of the severe crop pest whitefly species in cassava in two decades?

High populations of African cassava whitefly (Bemisia tabaci) have been associated with epidemics of two viral diseases in Eastern Africa. We investigated population dynamics and genetic patterns by comparing whiteflies collected on cassava in 1997, during the first whitefly upsurges in Uganda, with collections made in 2017 from the same locations. Nuclear markers and mtCOI barcoding sequences were used on 662 samples. The composition of the SSA1 population changed significantly over the 20-year period with the SSA1-SG2 percentage increasing from 0.9 to 48.6%. SSA1-SG1 and SSA1-SG2 clearly interbreed, confirming that they are a single biological species called SSA1. The whitefly species composition changed: in 1997, SSA1, SSA2 and B. afer were present; in 2017, no SSA2 was found. These data and those of other publications do not support the 'invader' hypothesis. Our evidence shows that no new species or new population were found in 20 years, instead, the distribution of already present genetic clusters composing SSA1 species have changed over time and that this may be in response to several factors including the introduction of new cassava varieties or climate changes. The practical implications are that cassava genotypes possessing both whitefly and disease resistances are needed urgently.