Cut, Root, and Grow: Simplifying Cassava Propagation to Scale.

Cassava (Manihot esculenta Crantz) is an essential crop with increasing importance for food supply and as raw material for industrial processing. The crop is vegetatively propagated through stem cuttings taken at the end of the growing cycle and its low multiplication rate and the high cost of stem transportation are detrimental to the increasing demand for high-quality cassava planting materials. Rapid multiplication of vegetative propagules of crops comprises tissue culture (TC) and semi-autotroph hydroponics (SAH) that provide cost-effective propagation of plant materials; however, they contrast the need for specific infrastructure, special media and substrates, and trained personnel. Traditional methods such as TC and SAH have shown promise in efficient plant material propagation. Nonetheless, these techniques necessitate specific infrastructure, specialized media and substrates, as well as trained personnel. Moreover, losses during the intermediate nursery and adaptation stages limit the overall effectiveness of these methods. Building upon an earlier report from Embrapa Brazil, which utilized mature buds from cassava for rapid propagation, we present a modified protocol that simplifies the process for wider adoption. Our method involves excising single nodes with attached leaves from immature (green) cassava stems at 2 months after planting (MAP). These nodes are then germinated in pure water, eliminating the need for specific growth substrates and additional treatments. After the initial phase, the rooted sprouts are transferred into soil within 1-8 weeks. The protocol demonstrates a high turnover rate at minimal costs. Due to its simplicity, cost-effectiveness, and robustness, this method holds significant promise as an efficient means of producing cassava planting materials to meet diverse agricultural needs.

Genetic complexity of cassava brown streak disease: insights from qPCR-based viral titer analysis and genome-wide association studies.

Cassava, a vital global food source, faces a threat from Cassava Brown Streak Disease (CBSD). CBSD results from two viruses: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). These viruses frequently pose challenges to the traditional symptom-based 1-5 phenotyping method due to its limitations in terms of accuracy and objectivity. Quantitative polymerase chain reaction (qPCR) offers precise virus quantification, although high costs hinder its widespread adoption. In this research, we utilized qPCR to measure the viral titer/load of CBSV and UCBSV. The objectives were to evaluate titer variability within the Cycle 2 (C2) population in two different environments, establish connections between viral titers and CBSD severity scores from the 1-5 scoring method, perform Genome-Wide Association Studies (GWAS) to identify genomic regions associated with CBSV and UCBSV titers, and investigate the functional annotated genes. The results demonstrated a significantly higher prevalence of CBSV (50.2%) in clones compared to UCBSV (12.9%) with mixed infections in some cases. Genotypic effects, particularly concerning UCBSV, were significant, with genotype-by-environment effects primarily influencing CBSV titer. GWAS Studies identified genomic regions associated with CBSV and UCBSV titers. Twenty-one SNP markers on chromosomes 10, 13, 17, and 18 exhibited significant associations with CBSV titer, collectively explaining 43.14% of the phenotypic variation. Additionally, 25 SNP markers on chromosomes 1, 2, 4, 5, 8, 11, 12, 13, 16, and 18 were associated with UCBSV titer, and explained 70.71% of the phenotypic variation. No shared genomic regions were identified between CBSV and UCBSV viral titers. Gene ontology analysis also revealed diverse gene functions, especially in transport and catalytic activities. These findings enhance our understanding of virus prevalence, genetics, and molecular functions in cassava plants, offering valuable insights for targeted breeding strategies.

Developing broad-spectrum resistance in cassava against viruses causing the cassava mosaic and the cassava brown streak diseases.

Growing cassava in Africa requires resistance against the viruses causing cassava mosaic disease (CMD) and the viruses causing cassava brown streak disease (CBSD). A dominant CMD2 resistance gene from a West African cassava landrace provides strong resistance against the cassava mosaic viruses. However, resistance against cassava brown streak viruses is limited to cassava varieties that show tolerance to the disease. A recently identified cassava germplasm that cannot be infected with cassava brown streak viruses provides a new source of the resistance required to protect cassava from CBSD. We present a synopsis of the status of virus resistance in cassava and report on the research to combine resistance against CBSD and CMD. We improve the lengthy and erratic screening for CBSD resistance by proposing a virus infection and screening protocol for the viruses causing CBSD and CMD, which allows a rapid and precise assessment of cassava resistance under controlled conditions. Using this approach, we classified the virus responses of cassava lines from Africa and South America and identified truly virus-resistant clones that cannot be infected with any of the known viruses causing CBSD even under the most stringent virus infections. A modification of this protocol was used to test seedlings from cassava crosses for resistance against both diseases. A broad-spectrum resistance was identified in a workflow that lasted 9 months from seed germination to the identification of virus resistance. The workflow we propose dramatically reduces the evaluation and selection time required in a classical breeding workflow to reach the advanced field trial stage in only 9 months by conducting selections for virus resistance and plant multiplication in parallel. However, it does not bypass field evaluations; cassava resistance assessment prior to the field limits the evaluation to candidates with virus resistance defined as the absence of symptoms and the absence of the virus. The transfer of our virus screening workflow to cassava breeding programs enhances the efficiency by which resistance against viruses can be selected. It provides a precise definition of the plant's resistance response and can be used as a model system to tackle resistance in cassava against other diseases.

Differential Tropism in Roots and Shoots of Resistant and Susceptible Cassava (Manihot esculenta Crantz) Infected by Cassava Brown Streak Viruses.

Cassava brown streak disease (CBSD) is a destructive disease of cassava in Eastern and Central Africa. Because there was no source of resistance in African varieties to provide complete protection against the viruses causing the disease, we searched in South American germplasm and identified cassava lines that did not become infected with the cassava brown streak viruses. These findings motivated further investigations into the mechanism of virus resistance. We used RNAscope® in situ hybridization to localize cassava brown streak virus in cassava germplasm lines that were highly resistant (DSC 167, immune) or that restricted virus infections to stems and roots only (DSC 260). We show that the resistance in those lines is not a restriction of long-distance movement but due to preventing virus unloading from the phloem into parenchyma cells for replication, thus restricting the virus to the phloem cells only. When DSC 167 and DSC 260 were compared for virus invasion, only a low CBSV signal was found in phloem tissue of DSC 167, indicating that there is no replication in this host, while the presence of intense hybridization signals in the phloem of DSC 260 provided evidence for virus replication in companion cells. In neither of the two lines studied was there evidence of virus replication outside the phloem tissues. Thus, we conclude that in resistant cassava lines, CBSV is confined to the phloem tissues only, in which virus replication can still take place or is arrested.

Duplex In Situ Hybridization of Virus Nucleic Acids in Plant Tissues Using RNAscope®.

RNAscope has been recently introduced by Advanced Cell Diagnostics (Newark, CA, USA) for in situ hybridization (ISH) of target RNAs using a proprietary technology for probe design and hybridization assay. The method has been extensively used as a basis for sensitive diagnostic assays in the medical field, while applications of this technique in plant sciences are still rare. Here, we describe a multiplex ISH protocol for detection of two plant viruses in formalin-fixed paraffin-embedded tissue sections from cassava. The dual-color protocol described can be used as reference for virus/host interaction studies including the visualization of virus nucleic acids and plant endogenous mRNAs. RNAscope provides a specificity and sensitivity of target detection that otherwise cannot be reached.

Resistance Against Cassava Brown Streak Viruses From Africa in Cassava Germplasm From South America.

Cassava brown streak disease (CBSD) is a severe virus disease of cassava and prevalent in the eastern regions of Africa. The disease is characterized by distinct vein chlorosis and streak symptoms on leaves and stems and necrosis of storage roots. This necrosis can encompass large areas of the root, rendering it inedible so that the entire cassava harvest can be lost. African cassava varieties are susceptible to either of the two viruses causing the disease, cassava brown streak virus (CBSV) and Uganda cassava brown streak virus, and while there are less sensitive varieties, all cassava eventually succumb to the disease. The lack of CBSD resistance in African cassava varieties prompted this search for new sources of virus resistance in the diversity of South American cassava germplasm held in the collection at International Center for Tropical Agriculture, Columbia. Our search for CBSD resistance in South American cassava germplasm accessions revealed that most of the 238 South American cassava lines infected with CBSV established systemic virus infections with moderate to severe disease symptoms on leaves and stems. Fifteen cassava accessions did not become virus infected, remained free of symptoms, and CBSV was undetected by qRT-PCR. When tuberous roots of those lines were examined, necrotic tissue was found in eight lines and CBSV was detected. The remaining seven cassava accessions remained clear of symptoms on all tissues and organs and were virus free. A broad spectrum of virus resistance also including other virus isolates was confirmed for the breeding lines DSC167 and DSC118. While detailed infection experiments with other cassava lines selected for resistance are still ongoing, this indicates that the resistance identified may also hold against a broader diversity of CBSVs. Taken together, we present the results of a comprehensive study on CBSV resistance and susceptibility in cassava germplasm accessions from South America. The virus resistance in cassava germplasm identified provides compelling evidence for the invaluable contribution of germplasm collections to supply the genetic resources for the improvement of our crops.

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.

Variability in P1 gene redefines phylogenetic relationships among cassava brown streak viruses.

BACKGROUND: Cassava brown streak disease is emerging as the most important viral disease of cassava in Africa, and is consequently a threat to food security. Two distinct species of the genus Ipomovirus (family Potyviridae) cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). To understand the evolutionary relationships among the viruses, 64 nucleotide sequences from the variable P1 gene from major cassava producing areas of east and central-southern Africa were determined. METHODS: We sequenced an amplicon of the P1 region of 31 isolates from Malawi and Tanzania. In addition to these, 33 previously reported sequences of virus isolates from Uganda, Kenya, Tanzania, Malawi and Mozambique were added to the analysis. RESULTS: Phylogenetic analyses revealed three major P1 clades of Cassava brown streak viruses (CBSVs): in addition to a clade of most CBSV and a clade containing all UCBSV, a novel, intermediate clade of CBSV isolates which has been tentatively called CBSV-Tanzania (CBSV-TZ). Virus isolates of the distinctive CBSV-TZ had nucleotide identities as low as 63.2 and 63.7% with other members of CBSV and UCBSV respectively. CONCLUSIONS: Grouping of P1 gene sequences indicated for distinct sub-populations of CBSV, but not UCBSV. Representatives of all three clades were found in both Tanzania and Malawi.