Efficient replication of cloned African cassava mosaic virus in cassava leaf disks

A transient viral replication assay for cloned African cassava mosaic virus (ACMV) was developed using cassava leaf disks. TMS60444 leaf disks were transfected using biolistic-mediated inoculation with ACMV clones pKACMVA and pKACMVB, which originate from West Kenya ACMV isolate 844 (ACMV-KE). Viral DNA synthesized de novo was monitored by Southern hybridization with an AV1 DNA probe. By using the methylation-sensitive restriction enzymes DpnI and MboI, it was possible to distinguish between the input DNA (dam-methylated) and the de novo synthesized viral DNA (not methylated). Different media used for pre- and post-culture of inoculated leaf disks significantly affected the efficiency of viral DNA accumulation. Without pre-culture, replicated viral DNA was not detectable. Culture time in optimized medium also affected the accumulation of nascent viral DNA, and the best results were obtained after 4 days pre-culture on CIM medium followed by 4-6 days post-culture in SH medium. Time-course analysis showed that viral DNA replication can persist for 5-6 days post-inoculation. Our results also confirmed that DNA B of ACMV could assist the accumulation of viral DNA in the leaf disks. The novel protocol described here has also been used successfully with other cassava cultivars (MCol22, MCol1505, TME282 and TMS92/0326) and ACMV clones from the ACMV Nigeria isolate (ACMV-NOg).