Dictyophara europaea (Hemiptera: Fulgoromorpha: Dictyopharidae): description of immatures, biology and host plant associations.

The European lantern fly Dictyophara europaea (Linnaeus, 1767), is a polyphagous dictyopharid planthopper of Auchenorrhyncha commonly found throughout the Palaearctic. Despite abundant data on its distribution range and reports on its role in the epidemiology of plant-pathogenic phytoplasmas (Flavescence dorée, FD-C), literature regarding the biology and host plants of this species is scarce. Therefore, the aims of our study were to investigate the seasonal occurrence, host plant associations, oviposition behaviour and immature stages of this widespread planthopper of economic importance. We performed a 3-year field study to observe the spatio-temporal distribution and feeding sources of D. europaea. The insects's reproductive strategy, nymphal molting and behaviour were observed under semi-field cage conditions. Measurement of the nymphal vertex length was used to determine the number of instars, and the combination of these data with body length, number of pronotal rows of sensory pits and body colour pattern enabled the discrimination of each instar. We provide data showing that D. europaea has five instars with one generation per year and that it overwinters in the egg stage. Furthermore, our study confirmed highly polyphagous feeding nature of D. europaea, for all instars and adults, as well as adult horizontal movement during the vegetation growing season to the temporarily preferred feeding plants where they aggregate during dry season. We found D. europaea adult aggregation in late summer on Clematis vitalba L. (Ranunculaceae), a reservoir plant of FD-C phytoplasma strain; however, this appears to be a consequence of forced migration due to drying of herbaceous vegetation rather than to a high preference of C. vitalba as a feeding plant. Detailed oviposition behaviour and a summary of the key discriminatory characteristics of the five instars are provided. Emphasis is placed on the economic importance of D. europaea because of its involvement in epidemiological cycles of phytoplasma-induced plant diseases.

First Report of Stolbur Phytoplasma Associated with Maize Redness Disease of Maize in Bosnia and Herzegovina.

Maize redness (MR), caused by stolbur phytoplasma (16SrXII-A, 'Candidatus phytoplasma solani') and vectored by the cixiid planthopper Reptalus panzeri (Löw), is a severe and emerging disease of maize in southeastern Europe (2). Symptoms of MR include midrib, leaf, and stalk reddening, followed by desiccation of the entire plant, abnormal ear development, and incomplete kernel set. MR may cause significant economic losses (2). During 2010, 2011, and 2012, the presence of MR-like symptoms on maize accompanied by significant yield losses were frequently observed in maize fields in the Semberija region of northeastern Bosnia and Herzegovina. From mid-June to early July, potential vectors were collected using mouth-aspirators from maize plants in fields at three locations in the Semberija region where MR-like symptoms were previously observed. At the end of July, symptomatic maize plants were collected from six fields in the same region for phytoplasma identification. In addition, we sampled asymptomatic johnsongrass (Sorghum halepense L.), bindweed (Convolvulus arvensis L.), and volunteer wheat (Triticum aestivum L.) in areas adjacent to maize fields with MR-like symptoms, as potential phytoplasma reservoirs (2,3). A total of 49 plants (38 maize, 6 johnsongrass, 3 bindweed, and 2 wheat) and 43 R. panzeri were tested for the presence of stolbur phytoplasma. Leaves of four maize seedlings, grown in insect-proof greenhouse conditions, were used as controls. Total DNA was extracted from roots of each plant and R. panzeri using the CTAB methods (2). Initial phytoplasma detection was conducted on 16S rRNA gene using nested PCR assay with phytoplasma universal primers P1/P7 and F2n/R2 (4). Subsequently, all phytoplasma positive samples were retested employing stolbur-specific Stol11 protocol with the f2r/f3r2 primer set (1). Molecular characterization of identified phytoplasmas was performed by PCR-RFLP analysis of the tuf gene (3) and by sequence analyses of the 16S rRNA nested PCR products (GenBank Accession No. KC852868). All samples that tested positive on 16S rRNA gene using phytoplasma generic primers gave positive reaction in assays with stolbur-specific primers. Stolbur phytoplasma was identified in 36 of 49 plant samples (34 of 38 symptomatic maize plants and in 2 of 6 johnsongrass) and in 2 of 43 R. panzeri individuals. None of the control plants, bindweed, or wheat samples were positive for the presence of any phytoplasma. Tuf gene RFLP analyses enabled affiliation of all isolates to the stolbur type tuf-b. Comparison of the 16S rRNA sequence using BLAST analyses further confirmed identification of the phytoplasmas as being 'Candidatus phytoplasma solani.' The obtained sequence showed 100% identity with 'Candidatus phytoplasma solani' from corn in Serbia (JQ730750). These data clearly demonstrated association of stolbur phytoplasma with MR symptoms on maize in Semberija, which represents the first report of the MR disease and stolbur phytoplasma in maize, R. panzeri, and johnsongrass in Bosnia and Herzegovina. In the Semberija region, maize-wheat crop rotation is a traditional practice, which is a key factor for MR occurrence and persistence (2). References: (1) D. Clair et al. Vitis 42:151, 2003. (2) J. Jovic et al. Phytopathology 99:1053, 2009. (3) M. Langer and M. Maixner. Vitis 43, 191, 2004. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

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 Alder Yellows Phytoplasma Infecting Common and Grey Alder (Alnus glutinosa and A. incana) in Montenegro.

Alder yellows phytoplasmas (AldYp) of the 16SrV-group associated with common alder (Alnus glutinosa) and grey alder (A. incana) are closely related to the grapevine yellows (GY)-associated quarantine phytoplasma Flavescence dorée (FDp). AldYp have been reported in several countries where epidemic appearance of FDp has been confirmed (France, Italy, and Serbia) (1,2). To date, the presence of 16SrV-group of phytoplasmas has not been reported in Montenegro; however, the main vector of FD phytoplasma, Scaphoideus titanus, has been identified in Montenegrin vineyards since 2008. During a survey in September 2011, in the northern part of Montenegro, 12 symptomatic alder trees showing symptoms of leaf discoloration, ranging from yellow to light green, were sampled. Six samples, each comprising several symptomatic leaves, were collected from A. glutinosa at streamside in woodlands near the town Kolasin and other six samples from A. incana close to the river Lim near the town of Bijelo Polje. Leaves of six young A. glutinosa seedlings were used as controls. Total DNA was extracted from fresh leaf midribs and petioles using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Nested PCR assay was conducted on 16S rRNA gene using phytoplasma generic primers P1/P7 and F2n/R2 followed by RFLP with MseI endonuclease (Fermentas, Vilnius, Lithuania) (3). Confirmation of identification and characterization of phytoplasma positive samples was performed by amplifying the non-ribosomal metionine aminopeptidase (map) gene using FD9f5/MAPr1 and FD9f6/MAPr2 primer set (1), specific for the members of the 16SrV group phytoplasmas. Amplification products were sequenced and deposited in GenBank (KC188998 through 9001). Comparison of the map gene sequences was performed by phylogenetic analysis along with 20 reference sequences of the 16SrV-group members (1), using the neighbor-joining method in MEGA5 software (4). 16S rRNA gene amplification revealed the presence of phytoplasmas in 11 out of 12 symptomatic samples, while Mse I restriction analysis and comparison with reference strains (AldYp and FDp from Serbia) enabled affiliation of detected phytoplasmas to the 16SrV-group. None of the controls were positive for any phytoplasma. Phylogenetic analysis of the Montenegrin AldYp map gene sequences revealed presence of four different strains clustering within the previously defined clusters of the 16SrV-group members (1). Three different strains associated with symptomatic A. glutinosa were identified and they clustered either within the FD1, FD2, or PGY-C cluster, while a single detected strain from A. incana proved to be identical with PGY-A isolate of AldY phytoplasma infecting grapevine in Germany (AM384892). To our knowledge, this is the first report of the association of 16SrV-group phytoplasmas with common and grey alder in Montenegro, as well as the first report of FD-related phytoplasmas in Montenegro. Since alder trees are considered as a possible natural reservoir of the FD phytoplasmas (1), the finding of alders naturally infected with strains related to the FDp (FD1 and FD2 clusters) indicate a possible threat of economic importance to the grape production in Montenegro, which should be addressed in further research. References: (1) G. Arnaud et al. Appl. Environ. Microbiol. 73:4001, 2007. (2) T. Cvrkovic et al. Plant Pathol. 57:773, 2008. (3) I-M. Lee et al. Int. J. Syst. Evol. Bacteriol. 48:1153, 1998. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

Stolbur phytoplasma transmission to maize by Reptalus panzeri and the disease cycle of maize redness in Serbia.

Maize redness (MR), induced by stolbur phytoplasma ('Candidatus Phytoplasma solani', subgroup 16SrXII-A), is characterized by midrib, leaf, and stalk reddening and abnormal ear development. MR has been reported from Serbia, Romania, and Bulgaria for 50 years, and recent epiphytotics reduced yields by 40 to 90% in South Banat District, Serbia. Potential vectors including leafhoppers and planthoppers in the order Hemiptera, suborder Auchenorrhyncha, were surveyed in MR-affected and low-MR-incidence fields, and 33 different species were identified. Only Reptalus panzeri populations displayed characteristics of a major MR vector. More R. panzeri individuals were present in MR-affected versus low-MR fields, higher populations were observed in maize plots than in field border areas, and peak population levels preceded the appearance of MR in late July. Stolbur phytoplasma was detected in 17% of R. panzeri adults using nested polymerase chain reaction but not in any other insects tested. Higher populations of R. panzeri nymphs were found on maize, Johnsongrass (Sorghum halepense), and wheat (Triticum aestivum) roots. Stolbur phytoplasma was detected in roots of these three plant species, as well as in R. panzeri L(3) and L(5) nymphs. When stolbur phytoplasma-infected R. panzeri L(3) nymphs were introduced into insect-free mesh cages containing healthy maize and wheat plants, 89 and 7%, respectively, became infected. These results suggest that the MR disease cycle in South Banat involves mid-July transmission of stolbur phytoplasma to maize by infected adult R. panzeri. The adult R. panzeri lay eggs on infected maize roots, and nymphs living on these roots acquire the phytoplasma from infected maize. The nymphs overwinter on the roots of wheat planted into maize fields in the autumn, allowing emergence of phytoplasma-infected vectors the following July.