First Report of Stolbur Phytoplasma Affecting Cichorium intybus in Serbia.

Chicory (Cichorium intybus, Asteraceae) is a typical Mediterranean plant indigenous to Europe, western Asia, Egypt, and North America (3). It is commonly consumed as a fresh vegetable in salads. In rural areas of Serbia it grows as a weed in crops, but it is used in folk medicine to treat skin disorders due to its antihepatotoxic activity (3). Methanol extracts of chicory leaves showed moderate antibacterial activity against enteric bacteria (3). A phytoplasma-like disease, expressed as proliferation of chicory shoots and flowers, was observed on wild plants for the first time in Obrenovac vicinity (44°40' N, 20°20' E) in July 2012. A flattening of the stem with a large number of filamentous leaves, contortion and abnormal growth of flowers on the stem (typical fasciation symptoms) were observed. Diseased plants did not produce seeds. Total DNA was extracted from the leaf midveins of 15 symptomatic and five symptomless plants (4). PCR amplification of 1.5-kb 16S rDNA fragment was performed using DreamTaq Green master mix (Thermo Scientific, Lithuania) and phytoplasma universal primer pairs P1/16S-Sr (1). Products of nested PCR (1.2 kb) were obtained using primer pair R16F2n/R2 (1). Both amplicons were detected in all diseased samples; however, DNA from symptomless samples yielded no amplicons. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 PCR products was performed in independent reactions using four endonucleases (AluI, TruI1, HhaI and HpaII). RFLP patterns from chicory samples were compared to those of Stolbur (STOL), Aster Yellows (AY), Flavescence Dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (1). All RFLP profiles from the chicory samples were identical to STOL reference strain, indicating that diseased chicory was affected by a phytoplasma that belongs to 'Candidatus Phytoplasma solani' (16SrXII-A group). The 16S rDNA sequence of representative sample from symptomatic plant (Vp4) was deposited under accession number KF661322 in NCBI GenBank. It showed 100% identity to KF263684.1 from Iranian peach, JQ730742.1 from Serbian valerian, and JQ730750 from Serbian corn, all belonging to the 'Ca. P. solani' taxon. Puna chicory disease on C. intybus associated with a subgroup 16SrV-B of phytoplasma was detected in China (2). This is the first report of the Stolbur phytoplasma associated with fasciation of C. intybus in Serbia and worldwide. References: (1) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 56:1593, 2006. (2) Z. N. Li et al. Can. J. Plant Pathol. 34:34, 2012. (3) J. Petrovic et al. Fitoterapia 75:737, 2004. (4) J. P. Prince. Phytopathology 83:1130, 1993.

First Report of Blueberry Reddening Disease in Serbia Associated with 16SrXII-A (Stolbur) Phytoplasma.

Blueberries (Vaccinium corymbosum) are among the healthiest fruits due to their high antioxidant content. The total growing area of blueberries in Serbia ranges from 80 to 90 ha. A phytoplasma-like disease was observed for the first time during July 2009 in three blueberry cultivars (Bluecrop, Duke, and Spartan) grown in central Serbia, locality Kopljare (44°20'10.9″ N, 20°38'39.3″ E). Symptoms of yellowing and reddening were observed on the upper leaves and proliferating shoots, similar to those already described on blueberries (4). There was uneven ripening of the fruits on affected plants. Incidence of affected plants within a single field was estimated to be greater than 20% in 2009 and 50% in 2010. Blueberry leaves, together with petioles, were collected during two seasons, 2009 and 2010, and six samples from diseased plants and one from symptomless plants from each cultivar, resulting in 42 samples in total. For phytoplasma detection, total DNA was extracted from the veins of symptomatic and asymptomatic leaves of V. corymbosum using the protocol of Angelini et al. (1). Universal oligonucleotide primers P1/P7 were used to amplify a 1.8-kb DNA fragment containing the 16S rRNA gene, the 16S-23S spacer region, and the 5' end of the 23S rRNA gene. Subsequently, a 1.2-kb fragment of the 16S rRNA gene was amplified by nested PCR with the R16F2n/R16R2 primers. Reactions were performed in a volume of 50 µl using Dream Taq Green master mix (Thermo Scientific, Lithuania). PCR reaction conditions were as reported (3), except for R16F2n/R2 primers set (annealing for 30 s at 58°C). PCR products were obtained only from the DNA of symptomatic plants. Fragments of 1.2 kb were further characterized by the PCR-RFLP analysis, using AluI, HpaII, HhaI, and Tru1I restriction enzymes (Thermo Scientific, Lithuania), as recommended by the manufacturer. The products of restriction enzyme digestion were separated by electrophoresis on 2.5% agarose gel. All R16F2n/R2 amplicons showed identical RFLP patterns corresponding to the profile of the Stolbur phytoplasma (subgroup 16SrXII-A). The results were confirmed by sequencing the nested PCR product from the representative strain Br1. The sequence was deposited in NCBI GenBank database under accession number KC960486. Phylogenetic analysis showed maximal similarities with SH1 isolate from Vitis vinifera, Jordan (KC835139.1), Bushehr (Iran) eggplant big bud phytoplasma (JX483703.1), BA strain isolated from insect in Italy (JQ868436.1), and also with several plants from Serbia: Arnica montana L. (JX891383.1), corn (JQ730750.1), Hypericum perforatum (JQ033928.1), tobacco (JQ730740.1), etc. In conclusion, our results demonstrate that leaf discoloration of V. corymbosum was associated with a phytoplasma belonging to the 16SrXII-A subgroup. The wild European blueberry (Vaccinium myrtillus L.) is already detected as a host plant of 16SrIII-F phytoplasma in Germany, North America, and Lithuania (4). The main vector of the Stolbur phytoplasma, Hyalesthes obsoletus Signoret, was already detected in Serbia (2). The first report of Stolbur phytoplasma occurrence on blueberry in Serbia is significant for the management of the pathogen spreading in blueberry fields. Since the cultivation of blueberry has a great economic potential in the region, it is important to identify emerging disease concerns in order to ensure sustainable production. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) J. Jovic et al. Phytopathology 99:1053, 2009. (3) S. Pavlovic et al. J. Med. Plants Res. 6:906, 2012. (4) D. Valiunas et al. J. Plant Pathol. 86:135, 2004.