Molecular Characterization of Stolbur Group Subgroup E (16SrXII-E) Phytoplasma Associated with Potatoes in China.

Abstract

Phytoplasmas have been reported from more than 70 plant species in China, most of which are from woody plants and very few are from potatoes (Solanum tuberosum). During the growing seasons of 2005 through 2011, potato disease surveys were conducted in seed and commercial fields in Yunnan Province and Inner Mongolia. Potato plants displayed symptoms of curled, yellowish and purplish leaves, shortened internodes, aerial tuber formation, and few small malformed underground tubers. Although the location of the fields surveyed each year varied, the disease seems to have become increasingly prevalent. In Yunnan, disease incidence was 5 to 24% in 2005 and 15 to 100% in 2010 and 2011. In Inner Mongolia, disease incidence in seed potato fields was 5 to 15% in 2006 and 25 to 50% in 2011. Total DNA was extracted from the leaves, stems, and roots of symptomatic and asymptomatic plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. A nested PCR was performed by using primer pair P1/P7 followed by R16F2n/R16R2 to detect the presence of phytoplasmas (1,3). An approximate 1.25-kb PCR product was amplified from symptomatic plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting the 1.2-kb amplicon singly with restriction enzymes AluI, BfaI, HhaI, HpaI, KpnI, MseI, and TaqI. Comparing the RFLP patterns of samples with previously published phytoplasma strains, the phytoplasmas matched patterns of the stolbur group, subgroup E (16SrXII-E) (1). In addition, the PCR product from P1/P7, diluted 1:30, was amplified by using primer pair P1A/P7A (2). The nested PCR product was cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by the Core Lab of the University of Alaska Fairbanks and GENEWIZ (South Plainfield, NJ). Nucleotide sequences (GenBank Accession No. EU293841) were analyzed by iPhyClassifier software (4), confirming the relationship of this phytoplasma to 'Candidatus Phytoplasma fragariae' with RFLP patterns identical to group 16SrXII-E. To our knowledge, this is the first molecular characterization of the stolbur group phytoplasmas associated with potato disease in China. The potato is becoming increasingly important in China. The impacts of stolbur on potato yield losses, disease distributions, and insect vectors are currently under investigation.