Single and Mixed Phytoplasma Infections in Phyllody- and Dwarf-Diseased Clover Plants in Lithuania

Naturally diseased plants of clover (Trifolium spp.) exhibiting symptoms of clover phyllody (virescence and phyllody of flowers) or of clover dwarf (abnormally small leaves, shortened internodes, proliferation of shoots, and dwarf growth habit) were observed in fields in Lithuania. Phytoplasma group-specific polymerase chain reactions (PCRs) and restriction fragment length polymorphism (RFLP) analysis of 16S rDNA revealed that the plants were infected by two mutually distinct phytoplasmas. Clover phyllody-diseased plants were infected by a subgroup 16SrI-C (subgroup I-C) phytoplasma (CPh-L) related to clover phyllody (CPh-C) phytoplasma in Canada. Clover dwarf-diseased plants were infected by both CPh-L and a phytoplasma (CYE-L) related to clover yellow edge (CYE-C) phytoplasma (subgroup 16SrIII-B = III-B) in Canada. A 1.8-kbp fragment of rRNA operon from CYE-L was amplified, cloned, and sequenced, and putative restriction sites mapped. This sequence shared high similarity (99.7%) with that of CYE-C and exhibited no differences from CYE-C in RFLP patterns of 16S rDNA; therefore, we tentatively classified CYE-L in subgroup 16SrIII-B (type strain, CYE = CYE-C phytoplasma) of the X-disease phytoplasma group. These findings extend the known geographical ranges of subgroup I-C and subgroup III-B taxa to the region of northern Europe including Lithuania and suggest a role of the subgroup III-B phytoplasma in clover dwarf disease.