Disease symptoms of severe shoot proliferation resembling phytoplasmal disease symptoms were observed in early spring of 2003 in hop (Humulus lupulus L.) plant cvs. Magnum and Marynka that were grown in a commercial farm in Poland. Proliferation of shoots has not been previously reported in hop plants. To detect the presence of phytoplasmas in hops, young shoots from four symptomatic (two cultivars) and two symptomless (‘Magnum’) plants were assayed for phytoplasma 16S rDNA using polymerase chain reaction (PCR). In addition, leaf samples from healthy Catharanthus roseus plants and plants experimentally infected with the reference strains of aster yellows phytoplasma (AY1, group 16SrI-B) or apple proliferation phytoplasma (AP, group 16SrX-A) were included for comparison. Amplifications were performed using the universal phytoplasma primer pair P1/P7 in an initial assay, and universal primer pairs fA/rA (1), Pc399/P1694, or R16F2n/R16R2 (2) and group specific primer pair R16(I)F1/R16(I)R1 (3) in a nested reaction. Specific products were obtained in direct PCR with the universal primer pairs P1/P7 only for the control samples of the reference strains AY and AP. No visible product was amplified by the direct PCR from samples obtained from hops and healthy periwinkle plants. However, in nested PCR with primer pairs P1/P7 followed by primer pairs fA/rA, R16F2n/R16R2, Pc399/P1694, or R16(I)F1/R16(I)R1, specific DNA bands were observed from naturally infected hop plants (both four symptomatic and two symptomless) tested. No amplification products were observed from healthy periwinkle plants. The specificity of PCR products (obtained with universal R16F2n/R16R2 primer pair) was confirmed by restriction fragment length polymorphism (RFLP) analysis using AluI, MseI, HhaI, and RsaI for enzymatic digestion. RFLP patterns of these rDNA fragments for samples of naturally infected hops and for AY1 reference strain were similar and were characteristic of phytoplasma 16SrI-B subgroup. To our knowledge, this is the first evidence that hop shoot proliferation disease is associated with natural infection by phytoplasmas. Furthermore, detection of phytoplasma in asymptomatic hops underscores the need to fully elucidate the etiological role of this pathogen in the disease.
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