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

Abstract

Cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), ranks among the top seven biological threats to global food security. The disease poses a significant threat to cassava production in East and Central Africa (ECA). In Uganda, overall CBSD incidence increased by c. 20% since it re‐emerged in 2004, and the disease persistently reduces cassava yields and storage root qualities. The spread of CBSD has been studied spatially in fields in different agroecologies. However, within‐host distribution and accumulation of CBSV and UCBSV in naturally infected cassava plants is unknown. Therefore, within‐host CBSV and UCBSV distribution was studied to correlate CBSD symptoms with virus titre in organs of infected cassava. Leaf, stem and storage root samples, with and without symptoms, were collected from 10 genotypes of field‐grown cassava. Presence of CBSV and UCBSV was detected by RT‐PCR and virus levels determined by qRT‐PCR. CBSV was present in 100% of CBSD samples with symptoms, with 45·3% positive for presence of both CBSV and UCBSV. Tolerant cassava genotypes were infected with CBSV alone and accumulated higher titre in roots than in aerial organs. Susceptible genotypes were co‐infected with CBSV and UCBSV and exhibited variation in virus titre in each organ. Across genotypes, virus titre was lowest in the youngest leaves and highest in mature non‐senescing leaves. This information provides insight into the relationship between CBSV, UCBSV and their cassava host, and is valuable for CBSD resistance breeding, epidemiology studies and CBSD control.

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.…”

“…Primers for DCL qRT-PCR are described in Supplementary Table 1. The transcript levels of DCLs were quantified using qRT-PCR as described by Ogwok et al (2015).…”

A Virus-Derived Stacked RNAi Construct Confers Robust Resistance to Cassava Brown Streak Disease

“…The farmer-preferred cultivar TME 204 possesses inherent resistance to CMD (Rabbi et al, 2014; Beyene et al, 2016) but is susceptible to CBSD (Ogwok et al, 2015). Transgenic TME 204 lines were genetically engineered with RNAi construct p5001 (Chauhan et al, 2015) with the goal of modifying this cultivar to generate TME 204 germplasm with robust resistance to CBSD and CMD.…”

Field Level RNAi-Mediated Resistance to Cassava Brown Streak Disease across Multiple Cropping Cycles and Diverse East African Agro-Ecological Locations

“…TME 14 infected with CBSD was planted around each plot to ensure maximum disease transmission to the test materials. Prior to planting, the “spreader” TME 14 plants were tested by RT-PCR (Ogwok et al, 2015) to ensure that they harbored both CBSV and UCBSV. After planting in the field, plants were watered daily for 1 week and then three times per week thereafter.…”

“…Genomic DNA was removed using the TURBO DNA-free Kit (Ambion, Houston, Texas) and the resulting RNA run on a denaturing agarose gel to check for quality. RNA was used for cDNA synthesis and amplification of 3′-viral genomic regions of about 1607 bp (CBSV) and 1670 bp (UCBSV) essentially as described by (Ogwok et al, 2015) using degenerate primer combinations CBSVF2 (5′-GGRCCATACATYAARTGGTT-3′) (Mohammed et al, 2012) and CBSVDR (5′- GGATATGGAGAAAGRKCTCC-3′) (Mbanzibwa et al, 2011). PCR products were cloned as described earlier (Ogwok et al, 2015) and sequenced using the Sanger sequencing method.…”