Subgroup 16SrI-C Research Articles

Purple coneflower with reddening and phyllody: a new host of clover phyllody phytoplasma

Echinacea purpurea plants showing yellowing/reddening and phyllody symptoms were observed in southern Bohemia. Transmission electron microscopy observations showed phytoplasmas in sieve cells of symptomatic plants, but not in healthy ones. Direct and nested polymerase chain reaction with phytoplasma specific primers and subsequent restriction fragment length polymorphism analyses of 16S rDNA as well as sequence analyses of the 16S–23S ribosomal operon (1789 bp) enabled the classification of the detected phytoplasmas in the aster yellows group, ribosomal subgroup 16SrI-C. Identical analyses of the transcription factor tuf gene and the ribosomal protein rpl22, rps3 genes were used for further classification, and revealed the affiliations of the phytoplasmas to the tufIC and rpIC subgroups, respectively. The presence of clover phyllody phytoplasmas in purple coneflowers is a new record for this species.

First Report of a Phytoplasma Affecting Tomato in Poland.

During 2006, two tomato plants exhibiting dwarfing, twisting of shoots and leaves, and virescence and phyllody of flowers were observed in a greenhouse in western Poland. Total genomic DNA was extracted from approximately 3.5 g of leaf midribs and petioles using the modified cetyltrimethylammoniumbromide (CTAB) buffer method (3). Direct PCR was done with universal phytoplasma primers P1/P7 for amplification of ribosomal 16S rDNA. The PCR product (1.8 kb) was diluted 1:30 with sterile distilled water and used as DNA template for nested PCR with primers R16F2n/R16R2 (1). The final product was an expected 1.2-kb rDNA fragment amplified from infected tomato tissues. The DNA extracted from a Vinca sp. infected with phytoplasma of 16SrI-B subgroup and from a healthy tomato plant were used as positive and negative assay controls, respectively. Restriction fragment length polymorphism analysis of the final PCR product (1.2 kb) using enzymes MseI, KpnI, AluI, HhaI, HpaII, RsaI, TaqI, and TruI (1) indicated that phytoplasma from the tomato belonged to the subgroup 16SrI-C (1,2). The sequence obtained from the final PCR product (746 bp) was deposited in the GenBank database under accession number EF164961. Multiple sequence alignments with sequences of phytoplasma available from GenBank were performed using ClustalW software. The analysis revealed that the Polish isolate was co-identical (100%) to two phytoplasma sequences (GenBank Accession Nos. AY839617 and DQ078304) that belong to 16SrI-C subgroup. To our knowledge, this is the first report of a phytoplasma affecting tomato plant in Poland.

Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999–2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.

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.