First Report of Group 16SrXII-A Phytoplasma Causing Stolbur Disease in Saponaria officinalis Plants in Serbia

Abstract

Saponaria officinalis L. (Caryophyllaceae; also known as bouncingbet or soapwort) is a perennial medicinal plant important for the pharmaceutical industry and used as an expectorant, alterative, laxative, and ointment for some skin diseases and arthritic conditions. S. officinalis plants with typical symptoms (23% in 2011 and 47% in 2012) of phytoplasma infection were observed in Pancevo plantation, Serbia. The symptoms appeared in May with leaves changing color from green to brown with severe reddening and necrosis. Severely diseased plants died. The infected plants had a significant reduction in biomass and quality. To investigate the presence of phytoplasma, total DNA was extracted from 10 symptomatic and four asymptomatic plants by a CTAB method. The nested PCR was carried out using phytoplasma-specific primer set P1/16S-SR followed by R16F2n/R16R2, targeting the 16S rRNA gene sequence of 1.5 and 1.2 kb in length, respectively. The amplicons of expected size were obtained from the symptomatic plants, but not from the asymptomatic plants. To obtain restriction fragment length polymorphism (RFLP) patterns, the R16F2n/R2 amplicons were digested with AluI, TruI1, HpaII, and HhaI endonucleases. The resulting patterns indicated that seven plants were infected by a Stolbur phytoplasma belonging to the 16SrXII-A subgroup, since it had the identical RFLP pattern as the STOL reference strain. The 1.2 kb nested PCR products of representative isolate Sap7 were purified using PCR purification kit (Fermentas, Vilnius, Lithuania) according to the recommended protocol and sequenced using facilities of IMGGI SeqService, Belgrade, Serbia. The obtained sequence was deposited in the NCBI database (GenBank Accession No. JX866951). The phytoplasma 16S rRNA gene sequence from Sap7 had a sequence identity of 97% with GenBank accessions GQ273961.1 (‘Euonymus japonicus’ phytoplasma), JX311953.1 (Candidatus Phytoplasma solani clone 5043), JQ412100.1 (Iranian alfalfa phytoplasma M21), and JN561702.1 (‘Convolvulus arvensis’ stolbur phytoplasma clone P1/P7-Conv2/2010-Bg). To our knowledge, this is the first report of a natural infection of S. officinalis by 16SrXII-A subgroup (Stolbur) phytoplasma in Serbia. As cited by Lee et al. (1), the 16SrI-M subgroup phytoplasma in S. officinalis sample was already detected in Lithuania by Valiunas (2). The identification of phytoplasma in the Pancevo plantation caused the intensification of our biological control tests and efforts to reduce the ecological and economic impacts of these phytoplasmas.