Effect of mosaic virus diseases on dry matter content and starch yield of five cassava (Manihot esculenta Crantz) accessions in Ghana

The effect of mosaic virus diseases on dry matter content and starch yield of five local accessions of cassava, “Ankrah”, “AW/17, “Tomfa”, “Dagarti” and “Tuaka” was evaluated. Tomfa showed the highest (95%) incidence of the disease, index of severity of symptoms for all plants (ISSAP) of 3.70, as well as, for diseased plants (ISSDP) (3.84) while Dagarti did not show any phenotypic expression of the disease throughout the study period. Most of the accessions displayed mosaic disease symptoms two months after planting but by the fifth month had fully recovered. However, polymerase chain reaction (PCR)-based testing at 12 months after planting revealed the presence of ACMV in all the accessions while EACMV was observed in Ankrah, Dagarti and AW/17. Mean tuber (fresh root weight) and starch yield at 12 months after planting (MAP) was significantly (P ≤ 0.05) high in Ankrah while percentage dry matter was significantly higher in Dagarti than the other accessions. A negative correlation between starch yield and cassava mosaic disease incidence implies that a high mosaic incidence particularly in the first three months results in lower tuber and starch yields.Keywords: Mosaic virus diseases, dry matter, starch yield, PCR, disease incidenceAfrican Journal of Biotechnology Vol. 12(27), pp. 4310-431

Multiple Origins and Regional Dispersal of Resistant dhps in African Plasmodium falciparum Malaria

Cally Roper and colleagues analyze the distribution of sulfadoxine resistance mutations and flanking microsatellite loci to trace the emergence and dispersal of drug-resistant Plasmodium falciparum malaria in Africa

Engineering Cassava Mosaic Disease (CMD) Resistance in a Ghanaian Cassava Cultivar

Cassava is an important staple crop for an estimated 800 million people worldwide. Cassava production in Africa is constrained by cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Both viral diseases cause annual yield losses estimated around $2.1 billion. CMD is caused by nine distinct virus species known as cassava mosaic geminiviruses (CMGs, genus: Begomovirus family: Geminiviridae). This disease is widespread across all cassava-growing regions in Africa. CBSD is caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) (genus Ipomovirus; family Potyviridae). Unlike CMD, CBSD is prevalent in low to mid altitude regions of Eastern and Central Africa. The necrotic lesions on storage roots, which make them unfit for consumption or processing, further exacerbate economic losses due to CBSD. Efforts aiming at controlling incidence of both CMD and CBSD in Africa have focused on screening for resistance in both cultivated and wild species of cassava and subsequent introgression of resistance into farmer-preferred cultivars. Three sources of resistance (CMD1, CMD2 and CMD3) have been identified for CMD and have been introgressed into local germplasm. Similarly, a number of CBSD “tolerant” varieties or clones such as “Kiroba”, “Kaleso” KBH 2006/18 and MM 06/0082 have been identified. However, high heterozygosity and outcrossing in cassava and the limited diversity of these sources of resistance challenge development of durable resistance to CMD and CBSD via conventional breeding approach. The main goal of this project is to engineer broad-spectrum resistance against cassava mosaic disease (CMD) into a farmer-preferred cassava cultivar in Ghana using expertise and tools developed at the Plant Biotechnology laboratory, ETH Zürich. In the first chapter, I present results characterizing genetic diversity of cassava mosaic geminiviruses (CMGs) identified in farmers’ fields in Ghana. Extending this characterization to include other CMG species identified elsewhere in Africa, highly conserved genomic regions were identified. CMGs have a high propensity for mutation and recombination that can influence their evolution. Besides, the relative contribution of natural selection in shaping genetic variation and thus evolution of geminiviruses remains poorly studied. Due to the high genetic variation present in CMGs, developing durable and broad-spectrum resistance in the field is challenging. Molecular characterization of CMG species in farmers’ fields in Ghana resulted in the identification of an isolate of East African cassava mosaic virus (EACMV), closely related to the EACMV-Kenya virus (EACMKV). This is the first report showing that an EACMKV species is associated with CMD in Ghana, which warrants attention as this could have epidemiological implications for control of CMD. Using current phylogenetic approaches that infer evolutionary rate variation at individual sites along protein-coding sequences, we identified the replication-associated protein (Rep/AC1) and the coat protein (CP/AV1) of CMGS to be evolving under negative selection. Evidence of strong negative selection in the Rep and CP genes of cassava mosaic geminiviruses makes them suitable targets for engineering resistance to geminiviruses in cassava. In the second chapter, I present results on screening for CBSD in four cassava-producing regions in Ghana. Furthermore, the response of eleven selected farmer-preferred cassava cultivars to mixed infections of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) isolates is reported. Although CBSD has not been reported in West Africa, reports in Burundi and Democratic Republic of Congo indicate a westward drift of the disease into West Africa. In this chapter, we confirmed the absence of CBSD in farmer fields in the four cassava-producing regions in Ghana. However, susceptibility of all eleven cultivars to both CBSV and UCBSV infection is alarming. It is expected that these results will encourage extensive screening for CBSD across Ghana and West Africa. Furthermore, screening of other local cassava cultivars could lead to identification of sources of CBSD resistance. The use of complementary approaches such as transgenic technology is encouraged to speed up development of CBSD-resistant cassava for farmers. In the third chapter, I present results on the induction of friable embryogenic calli (FEC) in eleven cassava cultivars from Ghana. FEC induction in cassava is highly genotype-dependent making it difficult to achieve in several African cassava cultivars. We successfully induced quality FEC tissues in ADI 001, a farmer-preferred cultivar. Using the standard protocol for cassava transformation developed in our laboratory, we successfully transformed and regenerated plantlets of ADI 001. The application of flow cytometry for routine assessment of FEC ploidy levels was carried out to ensure that quality FEC tissues are used for genetic transformation. Furthermore, over 40% of recovered lines had a single insertion of the transgene, making them suitable for downstream molecular characterization and confined field trials. In the fourth chapter, I present results showing development and successful transfer of CMD resistance into a farmer-preferred cassava cultivar, ADI 001. Using the RNAi-based approach that has achieved some success in developing resistance against both RNA and DNA viruses, we tested the single and multiple target approach for engineering resistance against mixed CMG infections present in cassava fields. Based on the genetic diversity study of CMGs identified in farmers’ fields, the replication-associated protein (AC1) and the movement protein (BC1) were selected as targets for silencing. Double targeting of AC1 and BC1 resulted in a shift in viral population in cuttings multiplied from scions inoculated with mixed CMG population. However, expression of high levels of hairpin-derived small RNAs homologous to only the AC1 gene sufficed efficient silencing of mixed CMG population. Near immunity to mixed CMG infection achieved in transgenic dsAC1 line-42 was maintained in cuttings multiplied from scions inoculated with field infected rootstocks. The work presented in this thesis demonstrates successful transfer of CMD resistance through transgenic technologies into a cassava cultivar preferred by farmers in Ghana. The identification of high levels of resistance against mixed CMG infection in this local cultivar, ADI 001 (dsAC1 line-42) encourages field screening to evaluate durability of resistance engineered. More importantly, transfer of transgenic technologies to laboratories in Africa such as the Biotechnology and Nuclear Agriculture Research Institute (BNARI) in Ghana through building of capacity is necessary to encourage and sustain adoption of transgenic technologies in Africa

Screening for Resistance in Farmer-Preferred Cassava Cultivars from Ghana to a Mixed Infection of CBSV and UCBSV

Cassava brown streak disease (CBSD) caused by the Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is a threat to cassava production in Africa. The potential spread of CBSD into West Africa is a cause for concern, therefore screening for resistance in farmer-preferred genotypes is crucial for effective control and management. We multiplied a selection of eleven cassava cultivars grown by farmers in Ghana to test their response to a mixed infection of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) isolates using a stringent top-cleft graft inoculation method. Virus titers were quantified in the inoculated scions and cuttings propagated from the inoculated scions to assess virus accumulation and recovery. All cultivars were susceptible to the mixed infection although their response and symptom development varied. In the propagated infected scions, CBSV accumulated at higher titers in leaves of eight of the eleven cultivars. Visual scoring of storage roots from six-month-old virus-inoculated plants revealed the absence of CBSD-associated necrosis symptoms and detectable titers of CBSVs in the cultivar, IFAD. Although all eleven cultivars supported the replication of CBSV and UCBSV in their leaves, the absence of virus replication and CBSD-associated symptoms in the roots of some cultivars could be used as criteria to rapidly advance durable CBSD tolerance using breeding and genetic engineering approaches

Screening for resistance in farmer-preferred cassava cultivars from ghana to a mixed infection of cbsv and ucbsv

Cassava brown streak disease (CBSD) caused by the Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is a threat to cassava production in Africa. The potential spread of CBSD into West Africa is a cause for concern, therefore screening for resistance in farmer-preferred genotypes is crucial for effective control and management. We multiplied a selection of eleven cassava cultivars grown by farmers in Ghana to test their response to a mixed infection of CBSV (TAZ-DES-01) and UCBSV (TAZ-DES-02) isolates using a stringent top-cleft graft inoculation method. Virus titers were quantified in the inoculated scions and cuttings propagated from the inoculated scions to assess virus accumulation and recovery. All cultivars were susceptible to the mixed infection although their response and symptom development varied. In the propagated infected scions, CBSV accumulated at higher titers in leaves of eight of the eleven cultivars. Visual scoring of storage roots from six-month-old virus-inoculated plants revealed the absence of CBSD-associated necrosis symptoms and detectable titers of CBSVs in the cultivar, IFAD. Although all eleven cultivars supported the replication of CBSV and UCBSV in their leaves, the absence of virus replication and CBSD-associated symptoms in the roots of some cultivars could be used as criteria to rapidly advance durable CBSD tolerance using breeding and genetic engineering approaches

Efficient Genetic Transformation and Regeneration of a Farmer-Preferred Cassava Cultivar From Ghana

Cassava is an important staple crop that provides food and income for about 700 million Africans. Cassava productivity in Africa is limited by viral diseases, mainly cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Genetic barriers such as high heterozygosity, allopolyploidy, poor seed set, and irregular flowering constrain the development of virus-resistant cassava varieties via conventional breeding. Genetic transformation represents a valuable tool to circumvent several challenges associated with the development of virus resistance and other valuable agronomic traits in cassava. The implementation of genetic transformation in many local African cultivars is limited either by the difficulty to produce friable embryogenic callus (FEC), low transformation, and/or regeneration efficiencies. Here, we report the successful induction of organized embryogenic structures (OES) in 11 farmer-preferred cultivars locally grown in Ghana. The production of high quality FEC from one local cultivar, ADI 001, facilitated its genetic transformation with high shoot regeneration and selection efficiency, comparable to the model cassava cultivar 60444. We show that using flow cytometry for analysis of nuclear ploidy in FEC tissues prior to genetic transformation ensures the selection of genetically uniform FEC tissue for transformation. The high percentage of single insertion events in transgenic lines indicates the suitability of the ADI 001 cultivar for the introduction of virus resistance and other useful agronomic traits into the farmer-preferred cassava germplasm in Ghana and Africa.ISSN:1664-462

Efficient Genetic Transformation and Regeneration of a Farmer-Preferred Cassava Cultivar From Ghana.

Cassava is an important staple crop that provides food and income for about 700 million Africans. Cassava productivity in Africa is limited by viral diseases, mainly cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). Genetic barriers such as high heterozygosity, allopolyploidy, poor seed set, and irregular flowering constrain the development of virus-resistant cassava varieties via conventional breeding. Genetic transformation represents a valuable tool to circumvent several challenges associated with the development of virus resistance and other valuable agronomic traits in cassava. The implementation of genetic transformation in many local African cultivars is limited either by the difficulty to produce friable embryogenic callus (FEC), low transformation, and/or regeneration efficiencies. Here, we report the successful induction of organized embryogenic structures (OES) in 11 farmer-preferred cultivars locally grown in Ghana. The production of high quality FEC from one local cultivar, ADI 001, facilitated its genetic transformation with high shoot regeneration and selection efficiency, comparable to the model cassava cultivar 60444. We show that using flow cytometry for analysis of nuclear ploidy in FEC tissues prior to genetic transformation ensures the selection of genetically uniform FEC tissue for transformation. The high percentage of single insertion events in transgenic lines indicates the suitability of the ADI 001 cultivar for the introduction of virus resistance and other useful agronomic traits into the farmer-preferred cassava germplasm in Ghana and Africa