1. Multiplex PCR for the simultaneous detection of cassava mosaic and brown streak viruses in cassava plants
1Abarshi M. M., 1Mohammed I. U., 2Kumar P. L. 3Legg J. P. and 1Maruthi M. N.
1Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, United Kingdom
International Institute of Tropical Agriculture (IITA), 2Nigeria and 3Tanzania.
Background
CBSVs were detected in combinations with CMBs, ACMV, EACMV and EACMV-UG using new primers CBSVF3 & CBSVR3 specific for CBSVs (Fig. 3A to E). Mixed infections of CBSV and CBSUV were also detected in a single tube duplex RT-PCR using CBSVF2, CBSVR7 and CBSVR8 primers (Fig. 3F).
Three new sets of primers were tested for their efficiency to detect CBSVs and compared with the old primers on field-collected cassava samples from Ukerewe and Kibaha in Tanzania. The new primers were twice as efficient in detecting CBSVs compared to old primers (Fig. 4).
The newly developed primers were used for detecting CBSVs from Ukerewe, Kibaha and coastal Kenya, which revealed the presence of CBSV and CBSUV in all locations in single and mixed infections (Fig. 5).
Duplex PCR for CMBs & CBSVs using new primers
Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important limiting factors for cassava production in sub-Saharan Africa (Fig. 1). CMD is caused by a number of ssDNA viruses - cassava mosaic begomoviruses (CMBs), and CBSD is caused by two RNA viruses - Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV). Diagnostic methods for CMD have long been available while such protocols for CBSD especially in mixed infections are either inadequate or non-existent. We addressed this knowledge gap by developing diagnostic protocols for the simultaneous detection of both viruses. In our studies, CBSVs were detected in combination with African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) isolates in a single tube RT-PCR reactions.
The CTAB method was used to extract total nucleic acids from plants dually infected with CBSD and CMD to obtain both RNA and DNA virus molecules. A number of parameters including different primers, primer and Mg concentrations and annealing temperatures were optimised. The primers CBSV10 & CBSV11 and CBSVF3 & CBSVR3 were used for the amplification of CBSV, while those used to amplify CMBs included degenerate primers (Deng A & Deng B) for CMBs and virus-specific primers for ACMV, EACMV and EACMV-UG.
RT-PCR protocols were developed for the simultaneous detection of CMBs and CBSVs from dually infected cassava plants. CBSVs were detected using the existing primers CBSV10 & CBSV11 in mixed infections with CMBs, EACMV and EACMV-UG (Fig. 2A, B & D) but not with ACMV (Fig. 2C). When new sets of ACMV-specific primers were used, they did amplify both ACMV and CBSVs (Fig. 2E & F).
Figure 1. Symptoms of CBSD (A) and CMD (B) on cassava leaves. B A A A D D E
Figure 3. Detection of CBSVs with ACMV, EACMV and EACMV-UG by duplex RT-PCR using new CBSV-specific primers CBSV F3 & CBSV R3
Comparison of the efficiency of new & old primers
Materials & methods
Multiplex PCR for CMBs & CBSVs using existing primers
Figure 4. Comparison of primer efficiency for detecting CBSVs
Distribution of CBSVs in eastern Africa
Figure 2. Detection of CBSVs together with ACMV, EACMV and EACMV-UG by duplex RT-PCR using existing CBSV-specific primers CBSV 10 & CBSV 11
Figure 5. Proportion of CBSV and CBSUV single & mixed infections in farmer fields in Tanzania and Kenya