First Report of a 16SrI-C Group Phytoplasma Associated With a Yellows-Type Disease Affecting Willow Plants in China

Abstract

In May of 2008, a phytoplasma-like disease was observed on willows (Salix babylonica Linn) grown in the Shaanxi Province. Affected plants showed yellowed leaves with green veins and dieback. Incidence of the disease was less than 10%. Samples were collected from 10 symptomatic and five asymptomatic willow plants from five different areas in Shaanxi Province. Total DNA was extracted from 0.5 g of leaf midrib and stem phloem tissue with a modified cetyltrimethylammoniumbromide (CTAB) method (3). Resulting DNA extracts were analyzed by a nested PCR assay using phytoplasma 16S rRNA gene primer pairs R16mF2/R16mR1 followed by R16F2n/R16R2 (1), which amplified a 1,452- and a 1,246-bp product, respectively. Sequences of amplicons were almost the same. Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with AluI, MseI, HhaI, HpaI, RsaI, HinfI, and TaqI endonucleases (2) indicated that all symptomatic plants were infected by a phytoplasma belonging to aster yellows group (16SrI) subgroup C (16SrI-C) 'Candidatus Phytoplasma asteris'. None of the symptomless plants tested positive. Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession No. FJ179166) confirmed the results on the basis of RFLP analyses. Subsequently, the presence of the phytoplasmas in symptomatic plants was also confirmed by transmission electron microscopy. To our knowledge, this is the first molecular evidence of the presence of a phytoplasma associated with a yellows-type disease of willows in northern China and its association with aster yellow group 16SrI, subgroup 16SrI-C.