Optimization of diagnostic RT-PCR protocols and sampling procedures for the reliable and cost-effective detection of Cassava brown streak virus

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Abstract

Sampling procedures and diagnostic protocols were optimized for accurate diagnosis of Cassava brown streak virus (CBSV) (genus Ipomovirus, family Potyviridae). A cetyl trimethyl ammonium bromide (CTAB) method was optimized for sample preparation from infected cassava plants and compared with the RNeasy plant mini kit (Qiagen) for sensitivity, reproducibility and costs. CBSV was detectable readily in total RNAs extracted using either method. The major difference between the two methods was in the cost of consumables, with the CTAB 10× cheaper (£0.53 = US$0.80 per sample) than the RNeasy method (£5.91 = US$8.86 per sample). A two-step RT-PCR (£1.34 = US$2.01 per sample), although less sensitive, was at least 3-times cheaper than a one-step RT-PCR (£4.48 = US$6.72). The two RT-PCR tests revealed consistently the presence of CBSV both in symptomatic and asymptomatic leaves and indicated that asymptomatic leaves can be used reliably for virus diagnosis. Depending on the accuracy required, sampling 100–400 plants per field is an appropriate recommendation for CBSD diagnosis, giving a 99.9% probability of detecting a disease incidence of 6.7–1.7%, respectively. CBSV was detected at 10−4-fold dilutions in composite sampling, indicating that the most efficient way to index many samples for CBSV will be to screen pooled samples. The diagnostic protocols described below are reliable and the most cost-effective methods available currently for detecting CBSV.

Introduction

Cassava (Manihot esculenta Crantz, family Euphorbiaceae), Africa's second most important food crop after maize, provides more than half of dietary calories for over half of both the rural and urban populations in sub-Saharan Africa. Africa produces more cassava than the rest of the world combined; production exceeds 104 million tonnes annually (FAO, 2009). Cassava is particularly popular among the poor for the ease of cultivation, low input requirement, tolerance to low rainfall and poor soils, and ease of propagation through stem cuttings. However, cassava cultivation in sub-Saharan Africa is affected severely by two important viral diseases: cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) (Thresh et al., 1994, Hillocks and Jennings, 2003, Thresh and Cooter, 2005, Legg et al., 2006).

CMD is distributed throughout the cassava-growing area of sub-Saharan Africa, whereas CBSD was confined until recently to coastal and lake shore areas of Malawi in eastern and southern Africa and at altitudes below 1000 metres above sea level (masl) (Storey, 1936, Nichols, 1950, Hillocks et al., 1999). CBSD is more damaging economically than CMD in the coastal zones from Kenya to the Zambezi River in Mozambique where both diseases occur (Hillocks, 1997, Hillocks et al., 2001, Hillocks et al., 2002), because in sensitive varieties CBSD causes dry necrotic rotting of tubers which, when severe, makes them unfit for consumption (Storey, 1936, Nichols, 1950, Hillocks et al., 1996). Recently, CBSD was reported at mid-altitude levels (above 1000 masl) in DR Congo (Mahungu et al., 2003), Uganda (Alicai et al., 2007), western Kenya and the Lake zone areas of Tanzania (Legg and Jeremiah, 2008). While the precise reasons for CBSD emergence are yet to be established, the disease has been shown to be highly damaging with 10–100% incidence that can result in up to 70% decrease in root weight of infected plants compared to healthy plants (Hillocks et al., 2001).

CBSD foliar symptoms vary greatly but are characterised mainly by leaf chlorosis in feathery patterns, appearing first along the margins of veins and later developing into chlorotic blotches (Storey, 1936, Nichols, 1950). However, CBSD symptoms are often masked in the field due to plants also being affected by cassava green mite (Mononychellus tanajoa), sooty mould (growing on the honeydew excreted by whiteflies) and CMD. Symptoms also vary with the variety, crop age and environmental conditions (Hillocks et al., 1999) and the tendency of cassava to shed older mature symptomatic leaves especially during prolonged dry periods further add to the complexity of disease identification.

Serological and/or molecular techniques have been developed in order to provide more reliable diagnosis without having to rely on variable disease symptoms. An antiserum was raised to purified CBSV from cassava, which detected readily the virus in Nicotiana benthamiana but failed to detect asymptomatic infections in cassava (Lennon et al., 1985, Sweetmore, 1994). More recently, a CBSV coat protein gene has been expressed and the resulting protein used for antisera production to develop a more reliable enzyme-linked immune-sorbent assay (ELISA) (Winter, 2009). The sensitivity and reliability of this antisera have yet to be reported. A sensitive reverse transcriptase polymerase chain reaction (RT-PCR) protocol was developed by Monger et al. (2001a) and confirmed the association of Cassava brown streak virus (CBSV), of the genus Ipomovirus, family Potyviridae with CBSD (Monger et al., 2001b). An isolate of CBSV (MLB3 from Tanzania) is now fully sequenced (Mbanzibwa et al., 2009a) and based on the comparison of CP gene sequences, two main CBSV strains have been identified (Mbanzibwa et al., 2009b). CBSV has also been shown to be transmitted from infected to healthy cassava plants by whiteflies (Bemisia tabaci Gennadius) (Maruthi et al., 2005).

Although a sensitive RT-PCR technique is available for CBSV diagnosis (Monger et al., 2001a), the reports described sample preparation methods only briefly. Parameters such as the selection of plant tissue for virus detection, especially in the absence of CBSD symptoms, and the association of stem and root symptoms with virus infection were not investigated. Commercial kits used commonly for sample preparation and RT-PCR are expensive and alternative cheaper methods are required to reduce the cost of testing cassava, a particularly important consideration for research laboratories in Africa. The main goal of this study was therefore, to optimize cost-effective diagnostic protocols and sampling procedures for the reliable detection of CBSV in cassava plants.

Section snippets

Virus isolate, detection and characterisation

Cassava plants of an unknown variety infected with CBSV were collected by R.J. Hillocks in farmers’ fields in Nampula, Mozambique in 2007 and maintained subsequently in the quarantine facilities of the Natural Resources Institute (NRI), UK. The virus was grafted onto variety Ebwanateraka, which was found to be highly susceptible to CBSV infections. Presence of CBSV was confirmed by observing symptom expression on leaves and by RT-PCR using CBSV10 (5′-ATCAGAATAGTGTGACTGCTG-3′) and CBSV11

Virus characterisation and detection in cassava leaf, stem and root tissues

The partial CP gene of CBSV isolate Nampula consisted of 914 bases, and the sequence of which has been deposited in the EMBL nucleotide database under the accession number FN423417. BLAST analysis of CP gene sequences indicated that the Nampula CBSV shared 93% nucleotide identity to each of five CBSV isolates (accessions each with over 500 nucleotide sequences in the database): Type A (accession number AY008442), Type C (AY008440), KBH1 (FJ821795), KBH2 (FJ821794), and an isolate with accession

Discussion

The RT-PCR protocol, first developed by Monger et al. (2001a) for CBSV, is the only means of detecting CBSV reliably in infected cassava plants. However, information is not available on the type of tissue for diagnostic purposes, especially on asymptomatic leaves from the top of the plant which are available readily for collection but do not always express symptoms. In this study we have shown that CBSV was detectable both in symptomatic and asymptomatic leaves from all parts of infected

Acknowledgements

We acknowledge the funding received from Bill and Melinda Gates Foundation under the Great Lakes Cassava Initiative project for part of this study. Messrs Abarshi and Mohammed were partly supported by the Kebbi State Government, Nigeria. Prof Mike Thresh and Dr Lava Kumar commented on the manuscript.

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