Southern blot hybridization analysis revealed that the extrachromosomal DNAs (EC-DNAs) associated with witches’ broom (VAC) and walnut witches’ broom phytoplasmas and various strains of the Italian clover phyllody phytoplasma (ICPh) were highly homologous among themselves but distinct from EC-DNAs of aster yellows related phytoplasmas occurring in the same insect and plant hosts and collected at the same site as the ICPh strains. The EC-DNAs of various strains of the ICPh differed significantly in number and size, more markedly among samples from different host plant species than among samples from the same host plant species. However, experiments on insect-mediated transmission suggested that the size variation is not associated with plant host specificity. Sequence analysis of cloned fragments revealed the presence of highly conserved ORFs (with substantially invariant putative translation products) but also the presence of regions rich in short direct and inverted repeats, which may be the cause of the size variations. The partial sequence of an EC-DNA associated with VAC encoding a putative replication-associated protein indicated their close phylogenetic relationship with geminiviruses.


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