Unexpected Genome Variability at Multiple Loci Suggests Cacao Swollen Shoot Virus Comprises Multiple, Divergent Molecular Variants

Cacao swollen shoot virus (CSSV) [Badnavirus, Caulimoviridae] causes swollen shoot disease of cacao (Theobroma cacao) L. in West Africa. Beginning in ~2002, tests once effective for detection of CSSV failed to detect virus in ~50-70% of symptomatic cacao plants in Ghana and Cote d'Ivoire, suggesting the possible emergence of an uncharacterized CSSV variant(s). Asymptomatic and symptomatic cacao and non-cacao plant samples from Cote d'Ivoire were subjected to polymerase chain reaction (PCR) amplification using eight different sequence-specific and/ or degenerate primer pairs with an expected size amplicon of 375-1100 base pairs. The frequency of PCR-amplification was variable, depending on sample-primer combination. Virus was not detectable in all symptomatic samples, despite characteristic CSSV-like symptoms, and several asymptomatic samples were CSSV-positive. Phylogenetic analysis using Maximum Likelihood of DNA sequences determined from the amplicons resulting from each primer pair resolved two to three groups, two that were closely related to previously reported CSSV isolates, and a third previously undescribed group. Based on the badnavirus species threshold at ≥ 80% pairwise nucleotide (nt) identity for the taxonomicallyinformative RT-RNase H region of the genome, analysis of a partial fragment corresponding to this locus resolved four CSSV groups, at 66-99% nt identity, among the PCR-amplifiable field isolates. Also, sequence analysis of as many as seven additional regions of the CSSV genome revealed extensive within-genome variability. These findings provide robust evidence for extensive genomic variation among multiple, divergent CSSV variants associated with swollen shoot disease symptoms in cacao in West Africa.