Detection and molecular characterisation of an alfalfa phytoplasma in Argentina that represents a new subgroup in the 16S rDNA Ash Yellows group(‘Candidatus Phytoplasma fraxini’)

Abstract

A phytoplasma infecting alfalfa crops was detected and characterised in Argentina, the Argentinean Alfalfa witches¢-broom (ArAWB) phytoplasma. Typical witches¢-broom symptoms were observed in diseased alfalfa plants from fields of the ?Cuyo? region in the Andean midwest. Pleomorphic bodies were observed by electron microscopy in sieve tubes of the diseased plants. The results obtained from the sequence homology, similarity coefficients derived from RFLP of the 16S rDNA and phylogenetic analysis led us to include this phytoplasma in the 16Sr VII (Ash Yellows) group. However, the ArAWB phytoplasma showed several differences when compared to other members of group 16Sr VII. The RFLP analysis of partial 16S rRNA gene of two ArAWB isolates, digested with 16 restriction enzymes, showed differences between the ArAWB and the reference strain (AshY1T) in six enzyme patterns. Restriction patterns unique for the group and an exclusive HinfI restriction site were found in the ArAWB phytoplasma rDNA. Moreover, the similarity coefficients (0.92?0.86) were lower than those obtained among other group members. The significant differences detected suggested that this phytoplasma belonged to a subgroup different from those described so far. We propose therefore, that the ArAWB phytoplasma should be included in a new VII-C subgroup, closely related to the EriWB phytoplasma (VII-B) described in Brazil.