Dianthus barbatus-A New Host of Stolbur Phytoplasma in Serbia.

Sweet William (Dianthus barbatus, Caryophyllaceae) is a biennial or short-lived perennial plant native to southern Europe, from the Pyrenees to the Carpathians and the Balkans. During the summers of 2012 and 2013, phytoplasma-like symptoms were observed on D. barbatus plants on a Serbian plantation (Pancevo, 44°51'49″ N, 20°39'33″ E, 80 m ASL). Only seven symptomatic plants were observed in the summer of 2012. Disease incidence in 2013 was estimated to be less than 1% but increased during 2014 to 4%. Affected plants, showing symptoms of leaf reddening, malformation, and proliferation; flower bud deficiency; and abnormal shoot production, were tested for phytoplasmas. Samples were collected from seven symptomatic and three symptomless plants each year (20 samples), and total nucleic acid was extracted from midrib tissue using a method that includes a phytoplasma enrichment step and DNA purification by chloroform/phenol (3). Oligonucleotide primers specific to the phytoplasma 16S to 23S rRNA intergenic spacer region were used in polymerase chain reaction (PCR) assays on DNA extracted from Sweet William plants (1,3). Using phytoplasma universal primer pairs P1/P7 and P1/16S-Sr, phytoplasma-specific 1.8- and 1.5-kb amplicons were obtained from four and six symptomatic plants collected in 2012 and 2013, respectively. Nested PCR with R16F2n/R2 primers yielded ~1.2-kb amplicons from DNAs of all symptomatic plants (1). No amplicon was generated in PCRs conducted with DNA templates from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of amplified 1.2-kb fragments was performed using four endonucleases (AluI, Tru1I, HhaI, and HpaII). Comparative analysis was done using RFLP patterns of Stolbur (Stol), Aster Yellows (AY), Flavescence Doree-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas. PCR-RFLP patterns from tested samples were identical to those of the Stol reference strain, indicating that diseased Sweet William was affected by phytoplasma belonging to the 16SrXII-A (Stolbur) group. The sequence of a 1.2-kb rDNA PCR product derived from sample Tk9 (deposited under accession number KM401436 in NCBI GenBank) showed the closest identity (100%) to those of Bulgarian corn (KF907506.1), Iranian 'Bois Noir' (KJ637208.1), and two Serbian phytoplasmas (KJ174507.1 from Calendula officinalis and KF614623.1 from Paeonia tenuifolia), all belonging to the 'Candidatus Phytoplasma solani' Stolbur subgroup. Previously, Aster Yellows Phytoplasma (16SrI) had been detected in two Dianthus species: D. barbatus (Sweet William) and D. caryophyllus (carnation) (2). This is the first record of the 16SrXII-A phytoplasma subgroup being associated with yellowing and reddening of D. barbatus in Serbia. The Stolbur phytoplasma occurrence on Sweet William is significant for the management of the disease in Serbia. References: (1) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) P. Northover et al. http://www.umanitoba.ca/faculties/afs/MAC_proceedings/proceedings/ 2007/Philip_Northover.pdf , 2007. (3) J. P. Prince et al. Phytopathology 83:1130, 1993.

First Report on Natural Infection of Paeonia tenuifolia by 'Candidatus Phytoplasma solani' in Serbia.

Peony (Paeonia tenuifolia L.) is a herbaceous perennial plant known for its beautiful and showy flowers. In Serbia it is native to the Deliblato Sands and is used as an ornamental and medicinal plant in folk medicine. This plant species has become a rarity and for that reason peony was introduced into a botanical collection near Backi Petrovac (northern Serbia), where it has been maintained since 1988. Reddening of lower leaves observed on 10% of plants (5 of 50) in the collection at flowering in May 2012 gradually progressed throughout affected plants by the seed maturation stage. Five leaves from each of three reddened and three symptomless plants were sampled at the end of July 2012. Total nucleic acid was extracted separately from individual leaves (30 samples) using the CTAB (cetyltrimethylammonium bromide) method (2). A nested PCR assay using universal primer pairs P1/P7, followed by R16F2n/R16R2 (4), amplified 16S rDNA fragments of 1.8 and 1.2 kb, respectively. DNA from all three reddened plants (15 samples) yielded 1.2-kb amplicons after nested PCRs. Restriction fragment length polymorphism (RFLP) patterns obtained by digestion of nested products with endonucleases AluI, TruI, HpaII, or HhaI (Thermo Scientific, Lithuania) (4) were identical to those of the STOL reference strain included for comparative purposes, indicating that symptoms were consistently associated with plant infection by 'Ca. Phytoplasma solani' (Stolbur) phytoplasma. The 16S rDNA amplicons from two peony plants (1.2 kb from B15 and 1.8 from B18) were sequenced (GenBank Accession No. KC960487 and KF614623, respectively). BLAST analysis revealed a 100% identity between the sequences and GenBank sequences of Stolbur phytoplasma, subgroup 16SrXII-A phytoplasma, previously detected in maize (JQ730750) in Serbia and red clover (EU814644.1) in the Czech Republic. Phytoplasma associated diseases of other species of the genus Paeonia (P. lactiflora Pall. and P. suffruticosa Andrews) have been described elsewhere. Disease symptoms on P. lactiflora from Chile were associated with the phytoplasma that belongs to the ribosomal subgroup 16SrVII-A ('Ca. Phytoplasma fraxini') (1). Also, Stolbur phytoplasma from the 16SrXII group was detected on P. suffruticosa plants in China, manifesting yellowing symptoms (3). To our knowledge, this is the first report of naturally occurring Stolbur phytoplasma disease of P. tenuifolia L. in Serbia. References: (1) N. Arismendi et al. Bull. Insectol. 64:S95, 2011. (2) X. Daire et al. Eur. J. Plant Pathol. 103:507, 1997. (3) Y. Gao et al. J. Phytopathol. 161:197, 2013. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.