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 Oidium neolycopersici on Greenhouse Tomatoes in Serbia.

In September 2011, tomato (Solanum lycopersicum L. 'Big Beef') plants showing typical symptoms of powdery mildew were collected in a greenhouse in the vicinity of Padinska Skela (District of City of Belgrade) in Serbia. Numerous circular, white colonies of powdery mildew were observed predominantly on the adaxial surface of the leaves, the petioles, and the stems. The foliage of infected plants turned yellow and necrotic, which was followed by rapid defoliation. Disease incidence was estimated by counting plants with powdery mildew symptoms in a random batch of 100 plants in four replicates and estimated to be extremely high, approaching 90%. Tomato plants ('Novosadski Jabucar') were inoculated with conidia released from diseased tomato leaves positioned above the tomato leaves and maintained at 25°C with a 14-h photoperiod. Healthy tomato plants from the same lot, which were not exposed to the conidia shower, were used as negative control. The first white fungal colonies appeared on the leaves of the inoculated plants within 4 to 7 days after inoculation, while no fungal growth was observed in the control plants. To determine the morphological characteristics of the pathogen, surface mycelium was removed with small strips of clear adhesive tape and examined using light microscopy. Microscopic observation revealed mycelium with lobed appressoria and hyaline, ellipsoid-ovoid or doliform conidia (32.5 to 47.5 × 17.5 to 25 µm) with no distinct firosin bodies and which produced sub-terminal germ tubes. Conidia were produced on the unbranched, erect conidiophores (82.5 to 150 µm) consisting of a cylindrical foot-cell followed by one to three short cells. No chasmothecia were found. On the basis of morphological characteristics, the pathogen was identified as Oidium neolycopersici (4), which was confirmed by internal transcribed spacer (ITS) sequence analysis. Total DNA was extracted directly from the whitish spots of superficial mycelium on the leaves with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. PCR amplification and sequencing were performed with primers ITS1F and ITS4 (1). The nucleotide sequence of the representative isolate 809-11 (Accession No. JQ619840) shared 100% identity with 16 O. neolycopersici isolates deposited in GenBank from different parts of the world. Tomato powdery mildew caused by O. neolycopersici is present in many European (4) and other countries around the world (3) and is becoming economically very important as majority of the tomato cultivars have shown to be susceptible (2). To our knowledge, this is the first report of O. neolycopersici in Serbia. Because tomato is a very popular and widely grown vegetable in Serbia, the presence of a new and potentially harmful disease could endanger greenhouse as well as open field tomato production. References: (1) J. H. Cunnington et al. Australas. Plant Pathol. 32:421, 2003. (2) T. Jankovics et al. Phytopathology 98:529, 2008. (3) H. Jones et al. Mol. Plant Pathol. 2:303, 2001. (4) L. Kiss et al. Mycol. Res. 105:684, 2001.