High throughput real‐time RT‐PCR assays for specific detection of cassava brown streak disease causal viruses, and their application to testing of planting material

Abstract

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is causing severe losses in cassava production in Kenya, Tanzania and Uganda. Two real-time RT-PCR assays based on TaqMan chemistry capable of detecting and distinguishing these two viruses are described. These assays were used to screen 493 cassava samples collected from western and coastal Kenya, the main cassava regions of Uganda and inland Tanzania. Both viruses were found in all three countries and across regions therein. Association of CBSD leaf symptom status with CBSV and UCBSV assay results was weak, confirming the need for a diagnostic assay. For leaf samples that were observed with CBSD-like leaf symptoms but shown as CBSV and UCBSV negative by the RT-PCR assay, deep sequencing using a Roche 454 GS-FLX was used to provide additional evidence for the absence of the viruses. The probability of the CBSD associated diagnostics detecting a single CBSV or UCBSV positive sample amongst other non-CBSD samples was modelled. The results of this study are discussed in the context of the application of diagnostics of CBSD-associated viruses under the Great Lakes Cassava Initiative and the need to minimize the risk of further spread of the viruses with cassava multiplication material. It is shown that high throughput testing undertaken at Fera of 300 cassava leaves taken from fields for seed multiplication, when analysed in pools of 10, has given a 95% probability of detecting 1% infected plants in the field.

Distribution and accumulation of cassava brown streak viruses within infected cassava (Manihot esculenta) plants

“…This indicates that presence of UCBSV does not seem to affect accumulation of CBSV RNA and implies the existence of cassava genotype‐specific resistance to infection with UCBSV. Presence of CBSV in all samples studied here shows this virus to be present at higher levels than previously reported at Namulonge (Uganda), in contrast to UCBSV (Mbanzibwa et al ., ; Winter et al ., ; Legg et al ., ; Adams et al ., ); this indicates that CBSV is replacing UCBSV as the more common pathogen at this location. Additional studies would be beneficial to determine if a similar pattern is occurring elsewhere in East Africa.…”

“…Variation was seen in the amount of viral RNA present in the leaf canopy with leaf age and correlated with CBSD symptom appearance (Table S2); this confirms previous reports (Moreno et al ., ; Adams et al ., ). In aerial tissues, across genotypes, the youngest symptomless leaves accumulated the lowest amount of viral RNA, senescing leaves also had low amounts, stems moderate amounts, but mature non‐senescing leaves with symptoms accumulated the highest amount of viral RNA (Table S2).…”

“…Prospective primer sets targeting unique sequences of 75–200 bp were tested for amplification. In addition to primer sequences for amplification of UCBSV and CBSV, primer sets for amplification of the reference genes cytochrome c oxidase ( COX ) (Adams et al ., ), ubiquitin 10 ( UBQ10 ), serine‐threonine phosphatase ( PP2A ) and GTP‐binding ( GTPb ) protein (Moreno et al ., ) used for data normalization, were tested. The annealing temperatures, PCR efficiencies and melt curve analysis of amplicons of the primer combinations UCBSVqF and UCBSVqR, CBSVqF and CBSVqR, COXF and COXR, and PP2AF and PP2AR (Table S1) were further optimized and used in subsequent analysis.…”

Differential response of cassava genotypes to infection by cassava mosaic geminiviruses

“…Sample DNA was standardized to a concentration of 4.5 ng/μl and 10 μl subjected to SYBR Green I chemistry as described by Ogwok et al (2015). Amplification was performed using the following conditions: 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. Cytochrome c oxidase (COX) (Adams et al, 2013) was used as reference gene. The primers used for this experiment are listed in Supplementary Table 1.…”

First report of Ugandan cassava brown streak virus on cassava in Democratic Republic of Congo

“…For verification of the identity of the causal organism, leaf samples of affected plants were collected, dried, and sent to the Food and Environment Research Agency in the UK for analysis by real-time PCR, as described by Adams et al (2012). It was confirmed that the leaves were positive for UCBSV and negative for CBSV (GenBank Accession No.…”