Real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for detection of cassava brown streak viruses.

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants.

Interactions between Bacillus thuringiensis and selected plant extracts for sustainable management of Phthorimaea absoluta.

Phthorimaea absoluta is a global constraint to tomato production and can cause up to 100% yield loss. Farmers heavily rely on synthetic pesticides to manage this pest. However, these pesticides are detrimental to human, animal, and environmental health. Therefore, exploring eco-friendly, sustainable Integrated Pest Management approaches, including biopesticides as potential alternatives, is of paramount importance. In this context, the present study (i) evaluated the efficacy of 10 Bacillus thuringiensis isolates, neem, garlic, and fenugreek; (ii) assessed the interactions between the most potent plant extracts and B. thuringiensis isolates, and (iii) evaluated the gut microbial diversity due to the treatments for the development of novel formulations against P. absoluta. Neem recorded the highest mortality of 93.79 ± 3.12% with an LT50 value of 1.21 ± 0.24 days, Bt HD263 induced 91.3 ± 3.68% mortality with LT50 of 2.63 ± 0.11 days, compared to both Bt 43 and fenugreek that caused < 50% mortality. Larval mortality was further enhanced to 99 ± 1.04% when Bt HD263 and neem were combined. Furthermore, the microbiome analyses showed that Klebsiella, Escherichia and Enterobacter had the highest abundance in all treatments with Klebsiella being the most abundant. In addition, a shift in the abundance of the bacterial genera due to the treatments was observed. Our findings showed that neem, garlic, and Bt HD263 could effectively control P. absoluta and be integrated into IPM programs after validation by field efficacy trials.

Distribution and Molecular Diversity of Whitefly Species Colonizing Cassava in Kenya.

The whitefly, Bemisia tabaci (Gennadium, Hemiptera) has been reported to transmit viruses that cause cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) in many parts of sub-Saharan Africa (SSA). Currently, there is limited information on the distribution, species and haplotype composition of the whitefly populations colonizing cassava in Kenya. A study was conducted in the major cassava growing regions of Kenya to address this gap. Analyses of mitochondrial DNA cytochrome oxidase 1 (mtCO1) sequences revealed the presence of four distinct whitefly species: Bemisia tabaci, Bemisia afer, Aleurodicus dispersus and Paraleyrodes bondari in Kenya. The B. tabaci haplotypes were further resolved into SSA1, SSA2 and Indian Ocean (IO) putative species. The SSA1 population had three haplogroups of SSA1-SG1, SSA-SG2 and SSA1-SG3. Application of KASP genotyping grouped the Bemisia tabaci into two haplogroups namely sub-Saharan Africa East and Southern Africa (SSA-ESA) and sub-Saharan Africa East and Central Africa (SSA-ECA). The study presents the first report of P. bondari (Bondar's nesting whitefly) on cassava in Kenya. Bemisia tabaci was widely distributed in all the major cassava growing regions in Kenya. The increased detection of different whitefly species on cassava and genetically diverse B. tabaci mitotypes indicates a significant influence on the dynamics of cassava virus epidemics in the field. The study highlights the need for continuous monitoring of invasive whitefly species population on cassava for timely application of management practices to reduce the impact of cassava viral diseases and prevent potential yield losses.