ABSTRACT
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.
Subject(s)
Manihot , Plant Diseases , Potyviridae , Recombinases , Manihot/virology , Plant Diseases/virology , Potyviridae/genetics , Potyviridae/isolation & purification , Recombinases/metabolism , RNA, Viral/genetics , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Plant Leaves/virology , Nucleic Acid Amplification Techniques/methods , Reverse Transcription , Sensitivity and Specificity , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
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.
