First Report of a Group 16SrI, Subgroup B, Phytoplasma in Diseased Epilobium hirsutum in the Region of Tallin, Estonia.

Abstract

Symptoms of phyllody of flowers and general plant yellowing indicating possible phytoplasma infection were observed in diseased plants of hairy willow-weed (Epilobium hirsutum L., family Onagraceae) growing in a meadow at Harku Village near Tallin, Estonia. DNA was extracted from diseased E. hirsutum using a Genomic DNA Purification Kit (Fermentas AB, Vilnius, Lithuania) and used as a template in nested polymerase chain reaction (PCR). Ribosomal (r) DNA was initially amplified in PCR primed by phytoplasma universal primer pair P1/P7 (4) and reamplified in PCR primed by nested primer pair 16SF2n/16SR2 (F2n/R2) (1) as previously described (2). Products of 1.8 kbp and 1.2 kbp were obtained in PCR primed P1/P7 and F2n/R2, respectively, from all four symptomatic plants examined. These data indicated that the diseased E. hirsutum plants were infected by a phytoplasma, termed epilobium phyllody (EpPh) phytoplasma. The 16S rDNA amplified in PCR primed by nested primer pair F2n/R2 was subjected to restriction fragment length polymorphism (RFLP) analysis using restriction endonucleases AluI, MseI, HpaI, HpaII, HhaI, RsaI, HinfI, and HaeIII (Fermentas AB). On the basis of the collective RFLP profiles, EpPh phytoplasma was classified in group 16SrI (aster yellows phytoplasma group), subgroup B (aster yellows phytoplasma subgroup), according to the phytoplasma classification scheme of Lee et al. (3). The 1.8-kbp rDNA product of P1/P7-primed PCR, which included 16S rDNA, 16S-23S intergenic spacer region, and the 5' -end of 23S rDNA, was cloned in Escherichia coli using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, Ca) according to manufacturer's instructions and sequenced. The sequence was deposited in the GenBank database as Accession No. AY101386. This nucleotide sequence shared 99.8% sequence similarity with a comparable rDNA sequence (GenBank Accession No. AF322644) of aster yellows phytoplasma AY1, a known subgroup 16SrI-B strain. The EpPh phytoplasma sequence was highly similar (99.9%) to operons rrnA (GenBank Accession No. AY102274) and rrnB (GenBank Accession No. AY102273) from Valeriana yellows (ValY) phytoplasma infecting Valeriana officinalis plants in Lithuania. ValY phytoplasma was found to exhibit rRNA interoperon sequence heterogeneity (D. Valiunas, unpublished data). To our knowledge, this is the first report to reveal E. hirsutum as a host of phytoplasma and to demonstrate the occurrence of a plant pathogenic mollicute in the northern Baltic region.