Screening cassava lines for resistance against viruses causing cassava brown streak disease (CBSD) is cumbersome because of the unpredictable and erratic virus infections in the slow plant infection processes that are frequently not associated with distinct leaf symptoms and because of the reliance on the assessment of root necrosis as an indicator of plant resistance/tolerance. The selection of resistant candidates thus extends over several growing cycles and is still associated with uncertainties about the stage of virus infection. To reduce the time for selection of resistant crosses and the uncertainties associated with field screening, we have developed a fast-forward virus screening workflow to assess cassava seedlings from crosses of cassava brown streak- and cassava mosaic virus-resistant parents. After passing through an intensive and precise virus infection routine, cassava seedlings that carried resistance against cassava brown streak and mosaic viruses were identified. Taking the results of 195 seedlings from 18 crossing families together, it became evident that resistance against the viruses causing CBSD is a dominant trait. The protocol developed for virus resistance screening in cassava can be readily adopted. It shifts resistance evaluation from the field to the nursery and replaces the erroneous and lengthy virus infection and screening process with a method of precision and speed.

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.

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. 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. 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.

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.

Cassava witches’ broom disease (CWBD) has emerged as a significant threat to cassava production in the Oiapoque region of Amapá, Brazil. Diseased plants exhibit stunted growth, vascular necrosis, abnormal shoot proliferation, and distinctive broom-like appearance. This study aimed to characterize the disease and identify its causal agent(s). The assessment by high-throughput sequencing of total nucleic acids (DNA and RNA) from affected cassava tissues revealed Rhizoctonia theobromae (syn. Ceratobasidium theobromae ) associated with CWBD. PCR-based detection using species-specific primers confirmed the presence of R. theobromae in 74% of symptomatic samples. Genetic diversity analysis based on the Ca2+/calmodulin-dependent protein kinase gene showed low variability among Brazilian isolates compared with Asian populations, suggesting their recent introduction. This first report of R. theobromae causing CWBD in Brazil follows a recent report of the disease from French Guiana and highlights the urgent need for effective measures to prevent the further spread of this emerging pathogen, which poses a deadly threat to cassava cultivation in the rainforest, a significant risk to cassava production in Brazil, and regions with similar eco-climatic conditions.

Reliable detection of Rhizoctonia (Ceratobasidium) theobromae in cassava leaves is essential for managing cassava witches’ broom disease (CWBD). However, low pathogen loads and heterogeneous colonization often result in false negatives in conventional PCR assays. In this study, we developed a species-specific TaqMan real-time PCR (qPCR) assay targeting the CaMK/CaMKL locus of R. theobromae . The assay was validated using field samples collected from indigenous communities in Oiapoque, Amapá, Brazil. It showed high amplification efficiency (90–110%; R² > 0.99) and increased detection by 40.5% compared to conventional PCR, enabling reliable detection in leaf tissues previously classified as negative. No amplification was observed in healthy or non-CWBD samples, confirming assay specificity. This method provides a sensitive and reliable tool for pathogen detection, supporting disease surveillance and germplasm indexing.