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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°51'49â³ N, 20°39'33â³ 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.
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Oil pumpkin (Cucurbita pepo L.) is commonly used for oil production, mainly in central and eastern Europe (1). In Serbia, it grows only in the north (Vojvodina Province), up to 1,500 ha. In June 2008, typical bacterial spot symptoms (dark green, water-soaked, transparent and greasy spots with yellow margins) were observed for the first time, cultivated at the experimental fields near Backi Petrovac. Since then, bacterial spots were regularly observed on oil pumpkin in the beginning of the growing seasons and during rainy weather, with disease incidence ranging from 5 to 20%. Bacteria isolated from 40 diseased leaves formed white, round, convex, and mucoid colonies on nutrient sucrose agar (NSA). Eight representative strains were aerobic, gram-negative, non-spore-forming rods. All strains produced fluorescent pigment and catalase. In levan-oxidase-potato rot-arginine dihydrolase-tobacco hypersensitivity (LOPAT) tests (3), they induced a hypersensitive reaction in tobacco leaves, did not cause soft rot of potato tubers, and were positive for levan and negative for oxidase and arginine dihydrolase. According to the LOPAT profile, they were classified in the Ia subgroup of pseudomonads (3). Strains hydrolyzed aesculin, but were unable to hydrolyze starch or reduce nitrates to nitrites. Negative reactions were obtained with hydrogen sulfide and indole. Reactions were identical to those of reference strain Pseudomonas syringae pv. syringae CFBP 1582, which was included in all biochemical, physiological, and molecular tests for comparison. To identify the pathogen, PCR and DNA sequencing were employed. Fragments of 752 bp for the syrB gene and 1,040 bp for the syrD gene were amplified from all strains, using B1/B2 and SyD1/SyD2 primer sets, respectively (2). The pathogenicity was tested on seeds and seedlings of oil pumpkin cv. Olinka. Strains were grown for 48 h on nutrient broth (NB) at 28°C and bacterial suspensions of ~108 CFU ml-1 were used for inoculations. Sterile water was used as negative control. Seeds (at the BBCH-1-0 stage) allowed to imbibe water were wounded by needle, immersed in the bacterial suspensions, and maintained in humid petri dishes to allow symptom development. The cotyledons of seedlings at the BBCH-10 stage were inoculated by hypodermic needle and potted plants were maintained at 25 ± 1°C and 75% relative humidity. Symptoms, including dark green, water-soaked spots, appeared 5 to 7 days after inoculation of both seeds and seedlings. The bacterium was re-isolated from spots of all seeds and seedlings tested, fulfilling Koch's postulates (the identity of re-isolated strains was confirmed by pathogenicity, morphology, and biochemical features). No symptoms were observed on controls. 16S rDNA amplicons obtained from representative strain Tk21 and re-isolated strain Tk21R with fD1/rD1 primers (4) were sequenced and deposited in GenBank under accession nos. KF305578 and KF735064, respectively. The sequences showed 100% similarity to each other and P. syringae pv. syringae from pepper (KC816630.1) (China), Ficus carica (JQ071937) (Serbia), and culture-collection ICMP:3023 (HM190217). On the basis of the symptoms, biochemical tests, and 16S rDNA sequence homology, the pathogen was identified as P. syringae pv. syringae. To our knowledge, this is the first report of P. syringae pv. syringae causing bacterial leaf spot on oil pumpkin in Serbia. References: (1) J. Berenji et al. Oil pumpkin Cucurbita pepo. Monography. IFVC, Novi Sad, 2011. (2) K. Gasic et al. Pestic. Phytomed. 27:219, 2012. (3) R. A. Lelliott et al. J. Appl. Bact. 29:470, 1966. (4) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.
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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.
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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.
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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.
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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°51'49â³ N, 20°39'33â³ 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.
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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°20'10.9â³ N, 20°38'39.3â³ 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.
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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.
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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.
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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°53'44.17â³N 19°33'11.62â³E, 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.
RESUMO
Food chain, food safety, and food-processing sectors face new challenges due to globalization of food chain and changes in the modern consumer preferences. In addition, gradually increasing microbial resistance, changes in climate, and human errors in food handling remain a pending barrier for the efficient global food safety management. Consequently, a need for development, validation, and implementation of rapid, sensitive, and accurate methods for assessment of food safety often termed as foodomics methods is required. Even though, the growing role of these high-throughput foodomic methods based on genomic, transcriptomic, proteomic, and metabolomic techniques has yet to be completely acknowledged by the regulatory agencies and bodies. The sensitivity and accuracy of these methods are superior to previously used standard analytical procedures and new methods are suitable to address a number of novel requirements posed by the food production sector and global food market.
Assuntos
Manipulação de Alimentos , Inocuidade dos Alimentos , Engenharia Genética , Proteômica , Animais , Humanos , Organismos Geneticamente ModificadosRESUMO
This paper documents the recovery of selected proteins from hepatic plasma membranes. Initial purification, achieved by a series of stepwise extractions, facilitates the subsequent purification by HPLC. Examples are provided to illustrate the recovery of specific proteins from two Morris hepatoma lines and the liver.
Assuntos
Neoplasias Hepáticas Experimentais/análise , Fígado/análise , Proteínas de Membrana/isolamento & purificação , Animais , Membrana Celular/análise , Cromatografia Líquida de Alta Pressão , Congelamento , Glicoproteínas/isolamento & purificação , Masculino , Peso Molecular , Ratos , Ratos Endogâmicos , Albumina Sérica/isolamento & purificação , SolubilidadeRESUMO
A series of hexa- to decapeptides (molecular mass range 800-1200) were labeled with naphthalene-2,3-dicarboxaldehyde, which preferentially reacts with the primary amino groups of a peptide. A highly stable peptide conjugate is formed, which allows selective analysis by fluorescence at excitation and emission wavelengths of 420 and 490 nm, respectively. After removal of unreacted compounds, the peptide conjugates were characterized by matrix-assisted laser desorption/ionization (MALDI) time-of-flight and nano-electrospray ionization (ESI) ion trap mass spectrometry. They readily form both [M + H]+ ions by MALDI and both [M + H]+ and [M + 2H]2+ ions by ESI. Furthermore, the fragmentation behavior of the N-terminally tagged peptides, exhibiting an uncharged N-terminus, was investigated applying post-source decay fragmentation with a curved field reflector and collision-induced dissociation with a quadrupole ion trap. Fragmentation is dominated in both cases by series of a-, b- and y-type ions and [M + H - HCN]+ ions. Peptide bonds adjacent to the fluorescence label were less susceptible to cleavage than the bonds of the non-derivatized peptide ions. In general, the resulting fragment ion patterns were less complex than those of the underivatized peptides.
Assuntos
Corantes Fluorescentes , Indóis/química , Espectrometria de Massas , Peptídeos/química , Espectrometria de Massas/métodosRESUMO
It was previously shown that vWF increases the rate of divalent cation-mediated fVIII reconstitution from isolated light chain (LCh) and heavy chain (HCh) subunits. We examined the effect of vWF on kinetic parameters for interaction between LCh and HCh in the presence of Ca2+ and Mn2+ ions, the most effective mediators of fVIII reconstitution from isolated subunits, and determined the minimal structural portion of vWF able to enhance fVIII formation. We found that affinity (Kd) for LCh/HCh binding mediated by Ca2+ and Mn2+ was 91 and 34.9 nM in the absence of vWF and 15.5 and 5.6 nM in its presence. This decrease of Kd resulted from a sixfold increase of the association rate constant (k(on)) for this interaction. The value of the dissociation rate constant (k(off)) for LCh/HCh complex was lower in the presence of Mn2+ (k(off) 4.6x 10(-6) s(-1)) than Ca2+ (k(off) 8.4 x 10(-6) s(-1)) but in both cases vWF had no effect on k(off). This indicates that at physiological concentration of 1 nM the rate of fVIII inactivation via dissociation to subunits would be entirely determined by the k(off) value, and it should not depend on the presence of vWF. Indeed, our experiments demonstrated that vWF did not have any effect on the rate of fVIII inactivation resulting from its dissociation to subunits at the physiological concentrations of the fVIII and vWF proteins. We identified the minimal portion of the vWF molecule, able to enhance reconstitution of fVIII from isolated subunits. Only vWF large proteolytic N-terminal homodimeric fragment SPIII (vWF residues 1-1365), but not small monomeric N-terminal fragment SPIII-T4 (1-272), both of which are known to contain a major fVIII binding site, was able to support reconstitution of fVIII activity from isolated LCh and HCh subunits in the presence of Mn2+ or Ca2+. The effect of SPIII on the LCh/HCh association was similar to that of vWF, because both proteins identically increased of the value of k(on) and did not alter the k(off) value.
Assuntos
Fator VIII/química , Fator VIII/metabolismo , Fragmentos de Peptídeos/farmacologia , Fator de von Willebrand/metabolismo , Fator de von Willebrand/farmacologia , Sítios de Ligação , Cálcio/farmacologia , Dimerização , Humanos , Cinética , Manganês/farmacologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de ProteínaRESUMO
We defined the main cause for the increased immunogenicity of the commercial factor VIII (fVIII) plasma-derived concentrates reported to induce formation of inhibitory antibodies in haemophilia A patients in Germany and Belgium. Formation of these antibodies directed against the C2 domain of fVIII was previously attributed to the use of solvent/detergent (S/D) treatment and pasteurisation for virus inactivation of fVIII concentrate. Since fVIII concentrates associated with increased immunogenicity were prepared from plasma pools characterised by elevated levels of coagulation markers, we examined whether the plasma source or S/D treatment and pasteurisation are responsible for structural changes within the C2 domain causing its abnormal immunogenicity. We found that samples of fVIII concentrate that originated from the abnormal plasma pool had a reduced ability to bind to phospholipid and conformationally sensitive anti-C2 domain antibodies, this effect being mostly pronounced in the samples that underwent both S/D treatment and pasteurisation. Thus, our study suggests that insufficient quality of the starting plasma pools is the major factor determining the structural alterations in the C2 domain of fVIII, whereas combination of S/D treatment and pasteurisation aggravates these changes.
Assuntos
Fator VIII/química , Antitrombina III/metabolismo , Detergentes/farmacologia , Fator VIII/efeitos dos fármacos , Fator VIII/imunologia , Fibrinogênio/metabolismo , Hemofilia A/complicações , Hemofilia A/tratamento farmacológico , Humanos , Peptídeo Hidrolases/metabolismo , Fosfolipídeos/metabolismo , Plasma/química , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Solventes/farmacologia , EsterilizaçãoRESUMO
The paper describes the production of a prothrombin complex concentrate (PCC) with high virus safety and a well-balanced content of vitamin K-dependent clotting factors and inhibitors. Solid-phase extraction is followed in a second step by optimized anion exchange chromatography using a radial column. A step for virus removal by nanofiltration is introduced in addition to the solvent/detergent step. By speeding up the chromatographic step, the period of time required for production is reduced considerably. The activities of the four vitamin K-dependent clotting factors II, VII, IX and X are in ratios of about 1:1:1:1. Protein C, Protein S, and Protein Z are also present in therapeutically effective concentrations. The product shows no thrombogenicity, in either in vivo nor in vitro models. Clinical investigations show that the PCC is a safe and efficient preparation for the substitutive treatment of FIX or FVII in patients suffering from the respective deficiencies. All bleeding episodes have been efficiently controlled with relatively low doses of the concentrate. The surgical procedures have been conducted without any problems in severely FIX and FVIII deficient patients.
Assuntos
Protrombina/isolamento & purificação , Animais , Humanos , Protrombina/farmacologia , Protrombina/uso terapêutico , Ratos , Trombose/prevenção & controleRESUMO
Dealing with the structural characterization of clotting factor VIII (FVIII) requires the application of several high-resolution analytical techniques. Besides the analytical point of view, a detailed knowledge of FVIII structure, production and therapeutic application is necessary. This review gives an overview of most of the currently applied analytical methods and how they deal with the complex analytical problem, investigating FVIII in a sample matrix containing large amounts of accompanying plasma proteins.
Assuntos
Fator VIII/química , HumanosRESUMO
A highly enriched preparation of human clotting factor IX was produced by a combination of adsorption chromatography, hydrophobic interaction chromatography and heparin affinity chromatography. The introduction of adsorption chromatography with a hydroxyaminopropyl support allows the capture step to be carried out directly from the cryoprecipitate-depleted plasma with a chromatographic column in flow-through mode. This replaces the batch procedure used until now. The other two chromatographic steps are designed in such a way that the eluate from the preceding step can be directly applied, without any intermediate treatment of the sample. This cuts the period of time required for the process by almost 50%, and increases the yield considerably. The isolated factor IX contains practically no contaminants and has a specific activity over 200 IU/mg of protein.
Assuntos
Cromatografia/métodos , Fator IX/isolamento & purificação , Adsorção , Cromatografia de Afinidade/métodos , Heparina , HumanosRESUMO
Clotting factor IX preparations from human plasma (pdFIX) have been characterized using electrophoretic methods like sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Factor IX prior to and after activation with factor XIa was separated by one- and two-dimensional polyacrylamide gel electrophoresis and on isoelectric focusing gels. The main differences between the band patterns of the two pdFIX preparations are due to their purity. Vitronectin was identified by immunological techniques as major accompanying plasma protein, separated from factor IX and characterized by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis.
Assuntos
Sangue , Fator IX/química , Sequência de Aminoácidos , Western Blotting , Cromatografia em Gel , Eletroforese em Gel Bidimensional , Humanos , Concentração de Íons de Hidrogênio , Focalização IsoelétricaRESUMO
For inactivation of lipid-enveloped viruses during the production of fresh frozen and lyophilized human plasma, the solvent-detergent method was applied. In this process, the solvent tri-n-butyl phosphate is removed by extraction with castor oil. The removal of the non-ionic detergent Triton X-100 is performed by solid-phase extraction using reversed-phase supports. For this purpose, different polymer- and silica-based supports were tested. The highest capacity for Triton X-100 was achieved with C18 silica gels. These supports can bind more than 0.1 ml of Triton X-100 per ml of support. None of the proteins, e.g., clotting factors, bind to the support and therefore they pass through the column and their biological activity is hardly affected. The determination of detergent during the production process was also studied. The application of special columns allowing direct sample injection was introduced. This is a simple method for the rapid in-process determination of Triton X-100 in human plasma by reversed-phase chromatography under isocratic conditions. Using the method developed here, less than 1.0 ppm of Triton X-100 can be detected in less than 12 min without any sample pretreatment.