ABSTRACT
Evening primrose (Oenothera biennis L.) is a biennial medicinal, edible, and ornamental plant species. It has attracted great interest for its seed oil that contains gamma linolenic acid, thus distinguishing this plant as a main commercial source of this essential fatty acid (4). This species has been grown as a permanent member of a medicinal plant collection established near Backi Petrovac (northern Serbia) for 22 years. The first disease symptoms were recognized as red spots on leaf rosette in July 2011, spreading gradually during vegetative growth and covering 1/3 to 1/2 of the leaf surface. Symptoms, observed on 16% of the plants (32 of 200) in the second half of May 2012 and on 23% (69 of 300) at the beginning of May 2013, appeared as reddening of lower leaves of flower-bearing stems. Affected plants exhibited stunted growth, while reddening spread over other leaves of flower-bearing stems. In severely affected plants, the flower-bearing stems were poorly developed, frequently forming witches' brooms. For that reason, 30 reddened and 20 symptomless leaves (2 leaves per plant) were sampled in both July 2012 and 2013 and total nucleic acids were extracted. Direct PCR assays were performed using phytoplasma universal primer pair P1/P7 (2) to amplify 1,800-bp fragments (the 16S rRNA gene, the 16S-23S intergenic spacer region, and a part of the 5' region of the 23S rRNA gene). PCR products were used in nested PCR with primers R16F2n/R2 (2) to amplify 1,200-bp fragments. The identification of phytoplasmas was done using RFLP (restriction fragments length polymorphisms) analyses of R16F2n/R2 amplicons digested with AluI, Kpn I, HpaII, TruI1, or HhaI endonucleases (Thermo Scientific, Lithuania) (2). RFLP patterns were identical to that of STOL reference strain of the 16SrXII-A subgroup, indicating that symptomatic plants were infected with phytoplasma (2). The 16S rDNA nucleotide sequence of representative strain E7 was deposited in GenBank under accession number KF850526. The BLASTn search showed 100% homology to an Iranian strain (KF263684.1) from peach and Serbian strains JQ730742.1 and JQ730750 from valerian and corn, respectively, all belonging to 'Candidatus Phytoplasma solani' (Stolbur). Sequencing data confirmed the association of Stolbur phytoplasma with affected O. biennis plants. It has already been reported that phytoplasma infection caused yellows disease of O. biennis (1). Also, the virescence of O. hookeri was associated with phytoplasma strain OAY from aster yellows (AY) group (subgroups 16SrI-B), and selected as the reference strain for the novel taxon 'Ca. P. asteris' (3). Here we provide the first report of naturally occurring Stolbur phytoplasma disease of O. biennis in Serbia. References: (1) S. F. Hwang et al. Z. Pflanzenkr. Pflanzenschutz 105:64, 1998. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (4) E. Small and P. M. Catling. Canadian Medicinal Crops. NRC Research Press, Ottawa, Ontario, Canada, 1999.
ABSTRACT
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.
