Absolute quantification of cassava brown streak virus mRNA by real-time qPCR

Rudolph R Shirima,Daniel G Maeda,Edward Kanju,Gloria Ceasar,Flora I Tibazarwa,James P Legg

doi:10.1016/j.jviromet.2017.03.003

Abstract

Cassava brown streak disease (CBSD) is the most important virus disease of cassava and a major food security threat in Africa. Yearly economic losses of up to $100 million USD have been attributed to CBSD. The lack of information on plant-virus interactions has restricted progress in breeding for CBSD resistance. Virus quantification is becoming a major tool for the quick and reliable assessment of plant host resistance. Therefore, a protocol for specific absolute quantification of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) was developed. CBSV and UCBSV coat protein (CP) specific standard templates: CBSV (pFer2, 826bp) and UCBSV (pUF1-R1-1, 732) respectively were generated and maintained in a TA cloning vector. These were used to construct standard curves using a TaqMan qPCR assay. Standard curves with acceptable amplification efficiencies (90–105%) and coefficients of determination (R2) greater than 0.99 were obtained. Infected cassava plants were sampled from a screenhouse and the field and used to validate this assay. Results obtained by testing several screenhouse and field samples revealed consistent absolute quantification assays for different CBSV and UCBSV isolates. This study presents the first protocol for absolute quantification of CBSVs and is expected to accelerate screening for CBSD resistance and hence breeding for CBSD resistance. The use of the method presented here should improve the clarity of virus quantification data as the results obtained are not influenced by varietal, host, seasonal or environmental conditions. Screening efficiency will also be greatly improved as there is no need for the use of reference genes consequently allowing for a larger number of samples to be analyzed. This will increase experimental precision in a timely and cost effective manner.

Highlights

Cassava brown streak disease (CBSD) is the most important virus disease of cassava and a major food security threat in Africa (Patil et al, 2015)
CBSD is known to be caused by two species of Ipomoviruses of the family Potyviridae: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV)
Plasmids were recovered, linearized and used to construct standard curves. These standard curves were used to optimize absolute quantification assays for CBSVs in nucleic acids extracted from CBSVs-infected cassava

Summary

Introduction

Cassava brown streak disease (CBSD) is the most important virus disease of cassava and a major food security threat in Africa (Patil et al, 2015). Yield and storage root quality reductions of up to 70–100% have been reported (Hillocks et al, 2001), resulting in annual economic losses of up to US$ 100 million (Manyong et al, 2012; Ndunguru et al, 2015). Recent reports show that CBSD is extending its range rapidly towards Central Africa and poses a major threat to cassava production in the region and neighbouring West African countries (Bigirimana et al, 2011; Legg et al, 2011; Mulimbi et al, 2012). Other approaches include dissemination of clean planting material through community participation (Legg et al, 2015) and phytosanitary practices (Hillocks and Jennings, 2003; Storey, 1936)

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Conclusion