First Report of Colletotrichum clavatum Causing Quince Anthracnose in Serbia.

Quince (Cydonia oblonga Mill.) tree is traditionally grown in Serbia. The fruits are used for compote, marmalade, and brandy production. In December 2012, quince fruits cv. Leskovacka with symptoms of postharvest anthracnose were collected in a storage facility in the area of Sabac, western Serbia. The symptoms were observed on fruits approximately 2 months after harvest. The incidence of the disease was about 3%, but the symptoms were severe. Affected fruits showed sunken, dark brown to black lesions with orange conidial masses produced in black acervuli. Small pieces (3 to 5 mm) of necrotic tissue were surface sterilized for 1 min in 1% NaOCl, washed twice with sterile distilled water, and placed on potato dextrose agar (PDA). Macroscopic and microscopic morphology characteristics of three isolates were observed after growth on PDA for 7 days at 25°C under a 12-h photoperiod. Fungal colonies developed white to gray dense aerial mycelium with orange conidial masses in the center of the colony. Conidia were hyaline, aseptate, clavate with rounded distal apices, 15.2 (12.8 to 16.8) × 4.5 (4.0 to 5.2) µm (mean L/W ratio = 3.3, n = 100). Morphological characteristics are consistent with the description of Colletotrichum clavatum (2). Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA region using ITS1/IT4 primers and ß-tubuline 2 gene using T1/T2 primers. The nucleotide sequences were deposited in GenBank (ITS Accession Nos. KF908866, KF908867, and KF908868; ß-tubuline 2 gene KF908869, KF908870, and KF908871). BLAST analyses of ITS and ß-tubuline 2 gene sequences showed that isolates from quince were 100% identical to other C. clavatum in GenBank (ITS JN121126, JN121130, JN121132, and JN121180; ß-tubuline 2 gene JN121213 to 17, JN121219, JN121228, JN121261 to 62, and JN121266 to 69), thus confirming the morphological identification. To fulfill Koch's postulates, asymptomatic fruits of quince cv. Leskovacka (five fruits per isolate) were surface sterilized with 70% ethanol, wounded with a sterile needle, and inoculated with 50 µl of a spore suspension (1 × 106 conidia/ml). Five control fruits were inoculated with 50 µl of sterile distilled water. The experiment was repeated twice. After 10 days of incubation in plastic containers, under high humidity (>90% RH) at 25°C, typical anthracnose symptoms developed on inoculated fruits, while control fruits remained symptomless. The isolates recovered from symptomatic fruits showed the same morphological features as original isolates. C. clavatum previously indicated as group B (3), or genetic group A4 within the C. acutatum sensu lato complex (4), is responsible for olive anthracnose in some Mediterranean countries (1,2), and has been reported as causal agent of anthracnose on a wide range of other hosts including woody and herbaceous plants, ornamentals, and fruit trees worldwide (4). To our knowledge, this is the first report of C. clavatum in Serbia, and the first report of quince anthracnose caused by this pathogen in Europe. Anthracnose caused by C. clavatum can endanger the production and storage of quince in the future, and may require investigation of new disease management practices to control this fungus. References: (1) S. O. Cacciola et al. J. Plant Pathol. 94:29, 2012. (2) R. Faedda et al. Phytopathol. Mediterr. 50:283, 2011. (3) R. Lardner et al. Mycol. Res. 103:275, 1999. (4) S. Sreenivasaprasad and P. Talhinhas. Mol. Plant Pathol. 6:361, 2005.

First Report of Cercospora apii, Causal Agent of Cercospora Early Blight of Celery, in Serbia.

Celery (Apium graveolens var. dulce) is a very important vegetable crop intensively cultivated in eastern and southern Serbia. During a field survey in August and September 2012, we observed symptoms similar to those of Cercospora early blight in eastern Serbia, with some of the affected fields showing up to 80% disease severity. The lesions on leaves were amphigenous, subcircular to angular and more or less confluent. Lesions enlarged and merged with age, followed by the development of necrotic area causing a continuous deterioration of the plant. Conidiophores arising from the stromata formed dense fascicles, sometimes appearing solitary, brown at the base, paler toward the apex, simple, straight to slightly curved, and rarely geniculate (dimensions 40 to 90 × 5 to 8 µm). Conidia were solitary, hyaline, at first cylindro-obclavate then acicular to acicular-obclavate, straight to slightly curved, subacute to obtuse at the apex, while truncated and thickened at the base (dimensions 45 to 160 × 4 to 5 µm), 5 to 13 septate. Based on the morphological features, we identified the pathogen as Cercospora apii Fresen. (2). In order to obtain monosporic isolates of the fungus, single conidia were cultivated on potato dextrose agar (PDA). To confirm the pathogenicity of the isolates, 5 mm-diameter mycelial plugs from the PDA plates were placed upside down on the adaxial leaf surface of 2-week-old celery seedlings of cv. Yuta. Control plants were inoculated with a sterile PDA plug. Three leaves per plant were disinfected with 70% ethanol, epidermis was scratched with a sterile needle to promote the infection, and inoculated. A total of 12 plants were inoculated with the mycelial plugs and 12 were used as control plants. Inoculated and control plants were kept in a moist chamber for 48 h and then transferred to a greenhouse at 25 ± 2°C. After 2 weeks, the first necrotic spots appeared on inoculated leaves, similar to the symptoms manifested in the field, while control plants remained symptomless. The pathogen was re-isolated and its identity was verified based on morphological and molecular features. To confirm the pathogen's identity, three isolates (CAC4-1, CAC24, and CAC30) were subjected to molecular identification based on the internal transcribed spacer region (ITS) using the ITS1/ITS4 universal primers (5), a partial calmodulin gene (CAL) using CAL-228F/CAL2Rd primers (1,4), and partial histone H3 gene (H3) using CYLH3F/CYLH3R primers (3). Sequences of the amplified regions were deposited in GenBank under accessions KJ210596 to KJ210604. The BLAST analyses of the ITS sequences revealed 100% identity with several Cercospora species (e.g., C. apii [JX143532], C. beticola [JX143556], and C. zebrina [KC172066]), while sequences of CAL and H3 showed 100% identity solely with sequences of C. apii (JX142794 and JX142548). Based on combined morphological and molecular data, the pathogen infecting celery was identified as C. apii, which to our knowledge represents the first report of the presence of the causal agent of Cercospora early blight disease in Serbia. References: (1) I. Carbone and L.M. Kohn. Mycologia 91:553, 1999. (2) P. W. Crous and U. Braun. CBS Biodivers. Ser. 1:1, 2003. (3) P. W. Crous et al. Stud. Mycol. 50:415, 2004. (4) J. Z. Groenewald. Stud. Mycol. 75:115, 2013. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

First Report of Cercospora carotae, Causal Agent of Cercospora Leaf Spot of Carrot, in Serbia.

Carrot (Daucus carota L. subsp. sativus [Hoffm.] Arcang.) is an important vegetable in Serbia, where it is grown on nearly 8,000 ha. In August 2012, ~1,500 ha of carrot fields were inspected in southern Backa in North Serbia. In nearly 40% of the fields, severe foliar and stem symptoms characteristic of cercospora leaf spot of carrot, caused by Cercospora carotae (Pass.) Solheim (3), were observed. Lesions on stems were oblong, elliptical, and more or less sunken, while those on the leaves were amphigenous, subcircular, light brown in the center, and surrounded by a dark brown margin. Conidiophores emerging from the lesions formed very loose tufts but sometimes were solitary. Conidiophores were simple and straight to subflexuous with a bulbous base (17 to 37 × 3 to 5 µm). Conidia were 58 to 102 × 2 to 4 µm, solitary, cylindrical to narrowly-obclavate, and hyaline to subhyaline with 2 to 6 septa. To obtain monosporial isolates, the conidia from one lesion were placed on water agar plates at 25°C in the dark for 24 h, after which single germinated conidia were selected and each placed on a petri dish containing potato dextrose agar (PDA). To confirm pathogenicity of three of the isolates, Koch's postulates were tested on carrot seedlings (3-true-leaf stage of growth) of a Nantes cultivar, SP-80, with 12 plants tested/isolate and 12 non-inoculated plants used as a control treatment. The leaves were atomized until runoff with the appropriate C. carotae spore suspension (104 conidia/ml sterilized water), while control plants were atomized with sterile water. All plants were then incubated in a dew chamber for 72 h, then transferred to a greenhouse at 25 ± 2°C. After 2 weeks, characteristic symptoms resembling those observed in the field developed on all inoculated plants; control plants were asymptomatic. The pathogen was re-isolated from all inoculated plants, and identity of the re-isolated fungi confirmed morphologically as described above, and molecularly as described below. The pathogenicity test was repeated with no significant differences in shape and size of lesions, or dimensions of conidiophores and conidia among isolates. To verify the pathogen identity molecularly, the 28S rDNA was amplified and sequenced using the V9G/LR5 primer set (2,4) as well as internal primers OR-A (5'-ATACCCGCTGAACTTAAGC-3') and 2R-C (5'-AAGTACTTTGGAAAGAG-3'); the ITS region of rDNA using the ITS1/ITS4 universal primers (5); and histone H3 gene (H3) using the CylH3F/CylH3R primers (1). The sequences for the three isolates were deposited in GenBank as Accession Numbers KF468808 to KF468810, KF941306 to KF941308, and KF941303 to KF941305 for the 28S rDNA, ITS and H3 regions, respectively. BLAST results for the ITS sequences indicated 94% similarity to the ITS sequence of an isolate of Pseudocercosporella capsellae (GU214662) and 92% similarity to the ITS sequence of an isolate of C. capsici (HQ700354). The H3 sequences shared 91% similarity with that of several Cercospora spp., e.g., C. apii (JX142548), C. beticola (AY752258), and C. capsici (JX142584), all of which shared the same amino acid sequence of the encoded H3 protein. Also, the 28S rDNA sequences had 99% similarity (identity of 318/319, with 0 gaps) with the single sequence of C. carotae available in GenBank (AY152628), which originated from Norway. This is, to our knowledge, the first report of C. carotae on carrot crops in Serbia as well as southeastern Europe. References: (1) P. W. Crous et al. Stud. Mycol. 50:415, 2004. (2) G. S. de Hoog and A. H. G. Gerrits van den Ende. Mycoses 41:183, 1998. (3) W. G. Solheim. Morphological studies of the genus Cercospora. University of Illinois, 1929. (4) R. Vilgalys and M. Hester. J. Bacteriol. 172:238, 1990. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

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 Brenneria nigrifluens as the Causal Agent of Shallow-Bark Canker on Walnut Trees (Juglans regia) in Serbia.

In late summer 2011, shallow, irregular cankers were observed on trunks and branches of non-chemically-treated walnut trees (Juglans regia L.) on a 30-year-old orchard in the region of Fruska Gora (Vojvodina, Serbia). Disease incidence was ~80% and yield loss was ~50%. For pathogen isolation, small pieces (~5 mm diameter) of wood tissue collected at the edge of the cankers were macerated in sterile distilled water and streaked onto nutrient agar with 5% sucrose. Plates were then incubated at 28°C for 2 days. The prevalent bacterial colonies and those similar in appearance to Brenneria nigrifluens (Wilson et al.) Hauben et al. were purified on nutrient agar (NA). Eight gram-negative, oxidasenegative, catalase-positive strains, showing oxidative and fermentative metabolism, were selected for further characterization. To identify the bacteria on a molecular basis, we analyzed the 16S rDNA and gyr B gene sequences. The 16S rDNA partial sequences of analyzed strains were amplified using the primers P0 (5'-GAGAGTTTGATCCTGGCTCAG-3') and P6 (5'-CTACGGCTACCTTGTTACGA-3') (3). Additionally, the gyr B gene sequences were generated with primers GyrB-F (5'-MGGCGGYAAGTTCGATGACAAYTC-3') and GyrB-R (5'-TRATBKCAGTCARACCTTCRCGSGC-3') (2). All amplicons were purified using the QIAquick PCR purification kit (QIAGEN) according to the manufacturer's instructions and sequenced by Macrogen Inc. (Seoul, South Korea) using the same primers used for amplification. The sequences were edited using FinchTV v.1.4.0, assembled using the Clustal W program integrated into MEGA5 software (4), and deposited in NCBI GenBank under accessions JX484738 to 40 for the 16S rDNA gene and KC571240 to 47 for the gyr B gene. The 1,359-bp 16S rDNA sequences obtained for the eight strains were compared to the reference 16S rDNA sequences retrieved from GenBank. BLAST analysis revealed 100% homology of Serbian strains with sequences of B. nigrifluens (Z96095 and FJ611884). The gyr B gene sequences of our strains were 100% homologous to the sequences of B. nigrifluens deposited in GenBank (JF311612 to 15). Pathogenicity of all strains was confirmed on young fruits by infiltration of bacterial suspensions (108 CFU ml-1 from a 48 h NA culture) with syringe into the mesocarp of walnut fruits and by stem infiltration with syringes without needles into branch wounds (1). Inoculated fruits were incubated in plastic boxes for 8 days at 20°C, 80 to 100% RH, with a 12-h photoperiod. Inoculated plants were maintained for 3 months at 22 to 28°C with continuous light and at 70 to 80% RH in plastic tunnels. Inoculated fruits developed bark canker symptoms at the inoculation sites, which became necrotic and released a reddish brown exudate. Necrotic lesions were observed on inoculated branches. B. nigrifluens was reisolated from the margins of necrotic fruit and stem tissue. Physiological and biochemical tests showed that strains grew at 36°C and did not produce arginine dihydrolase, H2S, indole, nitrate, nor a fluorescent pigment on King's B medium. They did not induce a hypersensitive reaction on tobacco leaves and did not hydrolyse gelatin and starch. They produced acid without gas from glucose, inositol, sorbitol, arabinose, and sucrose, but not from maltose and lactose (1). Results of pathogenicity and biochemical tests were also the same for reisolated strains. This is the first report of B. nigrifluens as the causal agent of shallow-bark canker on walnut trees in Serbia. References: (1) E. G. Biosca and M. M. López. J. Plant Pathol. 94:105, 2012. (2) P. Ferrente and M. Scotrichini. Plant Pathol. 59:954, 2010. (3) A. Grifoni et al. FEMS Microbiol. Lett. 127:85, 1995. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

[Segmental portal hypertension as a rare cause of gastric hemorrhage--case report]. / Segmentalna portalna hipertenzija kao retki uzrok krvavljenja iz zeluca--prikaz bolesnika.

Segmental portal hypertension is a rare pathologic condition, which produce gastric bleeding. Spleen vein thrombosis is more often caused by pancreatic disease (inflammations, tumors). Diagnosis is difficult to perform. Initial treatment is conservative. After successful conservative treatment early surgery should be planned. Unsuccessful conservative treatment indicates surgery. Splenectomy absolutely eliminates risk from rebleeding. Prognosis of these patients depends from etiology of pancreatic disease. We present a 46 old woman who successfully operated in our Department of Surgery due to massive upper gastrointestinal bleeding caused by isolated portal hypertension.