Phenolic composition and biological activities of geographically different type of propolis and black cottonwood resins against oral streptococci, vaginal microbiota and phytopathogenic Fusarium species.

AIMS: A multidisciplinary approach was used to compare phenolic composition, radical scavenging and antimicrobial activity of propolis samples from different geographical localities, and plant resin against various microorganisms. METHODS AND RESULTS: Using UHPLC-qqqMS quantitative analysis, 28 phenolic compounds were determined. Caffeic and p-coumaric acids were identified as main phenolic acids in poplar propolis samples, except samples from Russia (P6) and China (P7). Radical scavenging activity (applying DPPH spectrophotometric assay) showed the highest activity of Serbian (40·51%) and Chinese (53·21%) propolis samples. Broth microdilution method was used for the oral cavity, fungal phytopathogenic and human vaginal isolates which have been identified at a molecular level. The most sensitive bacterial isolates were Lactobacillus acidophilus (MIC of 0·03-0·13 mg ml-1 ) and the oral streptococci isolates (MIC values of 0·19-0·13 mg ml-1 ). The most sensitive fungal phytopathogenic isolate was Fusarium oxysporum (MIC 0·003 mg ml-1 ). All samples, except propolis from Serbia (P4) and Turkey (P5), showed a strong antifungal activity against Fusarium sporotrichioides, Fusarium subglutinans and Fusarium proliferatum. CONCLUSION: The results of various tests indicate good radical scavenging and antimicrobial activity against important human and plant pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: A detailed propolis analysis is important when proposing a preparation of new biological antimicrobial products which have a positive impact on human health and reduce antibacterial resistance.

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 of 16SrXII-A Subgroup Phytoplasma (Stolbur) Associated with Reddening of Oenothera biennis in Serbia.

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.

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.

The First Report of Stolbur Phytoplasma Associated with Phyllody of Calendula officinalis in Serbia.

Pot marigold (Calendula officinalis L.) is native to southern Europe. Compounds of marigold flowers exhibit anti-inflammatory, anti-tumor-promoting, and cytotoxic activities (4). In Serbia, pot marigold is cultivated as an important medicinal and ornamental plant. Typical phyllody, virescence, proliferation of axillary buds, and witches' broom symptoms were sporadically observed in 2011 in Pancevo plantation, Serbia (44°51'49″ N, 20°39'33″ E, 80 m above sea level). Until 2013, the number of uniformly distributed affected pot marigold plants reached 20% in the field. Due to the lack of seed production, profitability of the cultivation was seriously affected. Leaf samples from 10 symptomatic and 4 symptomless marigold plants were collected and total nucleic acid was extracted from midrib tissue (3). Direct PCR and nested PCR were carried out with primer pairs P1/16S-SR and R16F2n/R16R2n, respectively (3). Amplicons 1.5 and 1.2 kb in length, specific for the 16S rRNA gene, were amplified in all symptomatic plants. No PCR products were obtained when DNA isolated from symptomless plants was used. Restriction fragment length polymorphism (RFLP) patterns of the 1.2-kb fragments of 16S rDNA were determined by digestion with four endonucleases separately (TruI1, AluI, HpaII, and HhaI) and compared with those of Stolbur (Stol), Aster Yellows (AY), Flavescence dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (2). RFLP patterns from all symptomatic pot marigold plants were identical to the Stol pattern, indicating Stolbur phytoplasma presence in affected plants. The 1.2-kb amplicon of representative Nv8 strain was sequenced and the data were submitted to GenBank (accession no. KJ174507). BLASTn analysis of the sequence was compared with sequences available in GenBank, showing 100% identity with 16S rRNA gene of strains from Paeonia tenuifolia (KF614623) and corn (JQ730750) from Serbia, and peach (KF263684) from Iran. All of these are members of the 16SrXII 'Candidatus Phytoplasma solani' group, subgroup A (Stolbur). Phytoplasmas belonging to aster yellows (16SrI) (Italy and Canada) and peanut witches' broom related phytoplasma (16SrII) group (Iran) have been identified in diseased pot marigold plants (1). To our knowledge, this is the first report of natural infection of pot marigold by Stolbur phytoplasma in Serbia. References: (1) S. A. Esmailzadeh-Hosseini et al. Bull. Insectol. 64:S109, 2011. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) J. P. Prince. Phytopathology 83:1130, 1993. (4) M. Ukiya et al. J. Nat. Prod. 69:1692, 2006.

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

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. References: (1) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (2) D. Valiunas. PhD thesis, Institute of Botany, Vilnius, Lithuania, 2003.

First Report of Xanthomonas campestris pv. campestris as the Causal Agent of Black Rot on Oilseed Rape (Brassica napus) in Serbia.

In September 2010, leaves of oilseed rape (Brassica napus L.) with v-shaped, necrotic lesions on the leaf margins surrounded by yellow halos were collected. Symptoms were observed on the domestic cultivar Slavica (IFVC, Novi Sad) located in the Backa region, Vojvodina, Serbia, from a 3-ha field. Average disease incidence on 3-month-old plants was 45% (15 to 75%). Diseased leaves were rinsed in sterilized distilled water (SDW) and dried at room temperature for isolations. Leaf sections taken from the margin of necrotic leaf tissue were macerated in SDW and the extract was streaked onto yeast extract-dextrose-calcium carbonate (YDC) agar. Plates were incubated at 28°C for 3 days. Colonies were yellow, translucent, circular, and raised. Ten representative strains tested further were all gram-negative, catalase-positive, and oxidase-negative. The partial 16S rDNA sequence of a representative strain, TUr1, was amplified using primers fD1 and rD1 (2), and determined using the IMGGI SeqService facility in Belgrade. The 1,510-bp 16S rDNA sequence of TUr1 was compared to that of known strains in the NCBI GenBank database, and showed greatest similarity with that of Xanthomonas campestris pv. campestris (Xcc) strains ATCC 33913 and B100 (99% homology). Pathogenicity of 10 strains grown for 48 h on YDC at 28°C was completed using each of three methods: spraying a bacterial suspension (108 cfu/ml) onto the leaf surfaces of oilseed rape plants, stabbing the major veins of each of the first two true leaves with the tip of a sterile toothpick that had been dipped into a colony of the appropriate strain, and immersing cotyledons of the plants into a bacterial suspension (108 cfu/ml). All three tests were performed on 4-week-old oilseed rape plants of the cultivar Slavica. SDW was used for the negative control treatment for each method of inoculation. Reference strain Xcc NCPPB 1144 was used as a positive control treatment. Tests plants (two for each method of inoculation and each bacterial strain or control treatment) were maintained in a greenhouse at 25 ± 1°C and 80% relative humidity by keeping the plants in plastic bags. Two control plants for each of the negative and positive control treatments for each inoculation method were also enclosed in separate plastic bags. The bacterial strains and reference strain caused yellow lesions on inoculated plants that turned necrotic starting about 7 days after inoculation (DAI). The spots coalesced within 21 DAI to form necrotic areas. Plants inoculated with SDW remained symptomless. Reisolations were done onto YDC as described above. Reisolated strains showed the same colony morphology as described above. The bacterial strains grew at 35°C; produced levan from sucrose, hydrogen sulfide, and indole; did not reduce nitrate; hydrolyzed Tween 80; starch, gelatin, and aesculin; did not show tolerance to 0.10 and 0.02% triphenyl-tetrazolium chloride; and produced acid from d-arabinose, arginine, dulcitol, galactose, d-glucose, maltose, mannose, sorbitol, sucrose, and xylose (1). All strains tested by Plate Trapped Antigen-ELISAs (ADGEN Phytodiagnostics, Neogen Europe Ltd., Scotland) reacted with Xcc-specific polyclonal antibodies. Based on these tests, the strains were identified as Xcc. To our knowledge, this is the first report of this pathogen causing black rot of oilseed rape in Serbia. References: (1) T. B. Adhikariand and R. Basnyat. Eur. J. Plant Pathol. 105:303, 1999. (2) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

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.

First Report of 16SrIII-B Phytoplasma Subgroup Associated with Virescence of Arnica montana in Serbia.

Arnica montana L. is a high altitude perennial plant, indigenous to Europe, but it is not native in the flora of Serbia. Plantain introduction of A. montana began a few years ago in the Tara mountain region, Western Serbia (43°53'44.17″N 19°33'11.62″E, 1,008 m ASL). The mountainous climate in this region is characterized by 850 mm of precipitation per year and an average decade temperature range from 11 to 25°C in the vegetation period of May through September. The main soil type is dystric cambisol, exhibiting a slightly acidic reaction (pH 6.4). Seeds of A. montana cv. ARBO were obtained from the Agricultural Research Centre of Finland. Seedlings were produced in a greenhouse during the period March through April and planted in May 2008. Virescence symptoms were observed starting from May 2010. A. montana exhibited symptoms mainly on flowers, like green leaflike structures instead of flowers and proliferation of acillary buds. Later in the season, flowers were malformed and consequently failed to produce seeds. Plant material for analyses was collected during 2010 and 2011 from an experimental field located at Tara mountain. Total DNA was extracted from the leaf midveins of 14 symptomatic and six symptomless plants (3). Nested PCR was carried out with primers P1/P7 followed by P1/16S-Sr and R16F2n/R16R2 primers, resulting with the DNA fragments amplification of expected size: 1.8, 1.5, and 1.2 kb, respectively, in all symptomatic samples tested. No phytoplasmas were detected in symptomless samples. PCR products of 1.2 kb, obtained by R16F2n/R16R2 primers from symptomatic samples, were digested independently with four restriction enzymes (Alu I, Tru I, Hpa II and Hha I) and the RFLP patterns were compared with those of Stolbur (Stol), Aster Yellows (AY), Flavescence Doree-C (FD-C), Poinsettia Branch-Inducing (PoiBI) and Clover Yellow Edge (CYE) phytoplasmas (2). RFLP patterns from all symptomatic A. montana samples were identical to CYE pattern. Comparison of the 16S rDNA sequence of representative symptomatic sample Am4, deposited under accession number JX297491 in NCBI GenBank, with other phytoplasmas from the database revealed 99% identity with members of 16SrIII-B phytoplasma group: Clover yellow edge phytoplasma strain CYE (JQ944798.1), 'Euscelidius variegatus' phytoplasma strain AP-I (HQ589197.1), Clover phyllody phytoplasma strain CP (HQ589196.1), etc. In Serbia, phytoplasma belonging 16SrIII-B subgroup has been identified in Cirsium arvense (4) and pear plants (1). To our knowledge, this is the first report of a natural infection of A. montana by phytoplasma. Cultivation of A. montana provides the necessary raw material for medicament production. The flower heads are widely used for the topical treatment of bruises and sprains in phytopharmaceutical preparations.Taking into consideration that monoculture plantation growing of perennials favorites rapid spreading of infections, the present study tended to examine the potential threat of virescence, which could be the limiting factor of ex-situ conservation of this endangered plant by its cultivation. References: (1) B. Duduk et al. Acta Hortic. 781:351, 2008. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) J. P. Prince. Phytopathology 83:1130, 1993. (4) D. Rancic et al. Plant Pathology 54:561, 2005.