Specific Aim 1: Compare CMB evolution during vegetative propagation vs. whitefly transmission
Both vegetative propagation and whitefly transmission of CMB occur in the field but the extent to which transmission mode affects CMB evolution, as reported previously for other vector-borne viruses99, is not known. To address this question, we will inoculate virus-free cassava cuttings with infectious clones for ACMV and EACMV, which represent the major species groups of CMBs, either alone or in a mixed infection as the first step in parallel passaging experiments that move viruses to new plants through cuttings or by serial whitefly inoculation. Replicate lineages will be passaged 12 times at ca. 2-month intervals. NGS of rolling-circle-amplified (RCA) viral DNA at alternate passages will determine whether the mode of propagation influences viral genetic diversity over time. We will also monitor symptoms and virus titer, and collect whiteflies to determine CMB diversity in the vector (Aim 2) and leaf samples for small interfering RNA (siRNA) analysis (Aim 4). We will optimize many of the protocols and provide training for these experiments in pilot studies using a model begomovirus pathosystem.