A new 3D center of mass control approach for FES-assisted standing: First experimental evaluation with a humanoid robot.

This paper proposes a new control framework to restore the coordination between upper (functional) and lower (paralyzed) limbs in the context of functional electrical stimulation in completely paraplegic individuals. A kinematic decoupling between the lower and upper limbs controls the 3D whole-body center of mass location and the relative foot positions by acting only on the lower-limb joints. The upper limbs are free to move under voluntary control, and are seen as a perturbation for the lower limbs. An experimental validation of this paradigm using a humanoid robot demonstrates the real-time applicability and robustness of the method. Different scenarios mimicking the motion of a healthy subject are investigated. The proposed method can maintain bipedal balance and track the desired center of mass trajectories under movement disturbances of the upper limbs with an error inferior to 0.01 m under any conditions.

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 Alder Yellows Phytoplasma Associated with Common Alder (Alnus glutinosa) in the Republic of Macedonia.

Alder yellows phytoplasma (AldYp) is classified as a member of the 16SrV-group of phytoplasmas and is closely related to Flavescence dorée (FD), a quarantined pathogen of economic importance affecting vineyards across Europe. AldYp is associated with common (Alnus glutinosa) and grey alder (A. incana), and has been reported in France, Italy, Germany, Austria, Switzerland, the Baltic region, Serbia, and Montenegro (1,2,4). For Macedonian vineyards, so far, neither infection of grapevine with 16SrV-group of phytoplasmas nor the presence of the main FD phytoplasma vector, Scaphoideus titanus, has been recorded. However, the presence of FD-related phytoplasma was detected in wild Clematis vitalba. In September and October 2013, leaves with petioles from A. glutinosa exhibiting leaf discoloration and yellowing were collected from two sites (41°23'43″ N, 22°54' E and 41°23' N, 22°53' E) in southeast Macedonia near the village of Smolare (Strumica district). Eight samples were collected from each site. Leaves of six asymptomatic alder seedlings collected from the same sites served as a control. Nucleic acids were extracted from fresh leaf midribs and petioles using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Initial phytoplasma identification was carried out by nested PCR assay of the 16S rRNA gene, using universal primers P1/P7 and R16F2n/R16R2 followed by RFLP with MseI endonuclease (Fermentas, Vilnius, Lithuania), as previously reported (4). Characterization of detected phytoplasmas was performed by amplifying two genetic loci specific for the members of the 16SrV group phytoplasmas; the ribosomal protein gene operon (rp) using primers rp(V)F1/rpR1 and rp(V)F1A/rp(V)R1A (3), and the non-ribosomal metionine aminopeptidase (map) gene using primer set FD9f5/MAPr1 and FD9f6/MAPr2 (1). The PCR amplicons were sequenced and deposited in NCBI GenBank database under the accession numbers KJ605448 to 52 (map) and KJ605453 to 57 (rp). The obtained sequences were compared with reference sequences of the 16SrV-group phytoplasmas (1,3) using the neighbor-joining method in MEGA5 (5). The presence of phytoplasma was detected in 14 of 16 symptomatic alder samples, while all control plants tested negative. The MseI restriction profiles were identical among all 14 samples and with the reference strains of the 16SrV group phytoplasmas (EY1 - 16SrV-A, FD-C - 16SrV-C, and FD-D - 16SrV-D). The rp-based phylogeny enabled identification of four diverse phytoplasma strains among the AldYp strains from Macedonia. Three strains clustered within the rpV-E subgroup while one belonged to rpV-L subgroup. Phylogenetic analysis of the more variable genetic locus, map, showed the presence of five diverse phytoplasma strains. Four strains belonged to the map-FD2 (FD-D, FD92) cluster, while one grouped within the map-FD1 (FD70) cluster. To our knowledge, this is the first report of 16SrV phytoplasma group occurrence on alder in Macedonia. The significant similarity between AldYp strains and FD sensu stricto indicate the risk of pathogen exchange between the wild ecosystem and the grapevine (1). Alder trees naturally infected with the FDp-related strains could therefore represent a serious risk for FD outbreak in Macedonian vineyards if local S. titanus populations developed. References: (1) G. Arnaud et al. Appl. Environ. Microbiol. 73:4001, 2007. (2) T. Cvrkovic et al. Plant Pathol. 57:773, 2008. (3) M. Martini et al. Int. J. Syst. Evol. Microbiol. 57:2037, 2007. (4) S. Radonjic et al. Plant Dis. 97:686, 2013. (5) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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.

Quantitative evaluation of stromal myofibroblasts and their significance for the metastatic capacity of colorectal carcinoma.

PURPOSE: To quantify the myofibroblasts in the tumor stroma of colorectal carcinomas using immunostaining with anti smooth muscle actin (SMA) as a marker for myofibroblasts. METHODS: The study was carried out on 46 surgically resected primary colorectal adenocarcinomas from the archive of the Centre for Pathology and Forensic Medicine of the Military Medical Academy in Belgrade, from 2008-2010. All samples were analysed by the scientific software "Image J". Myofibroblasts were visualized using anti-SMA antibody and quantified in order to predict tumor capacity for invasion and metastasis. Receiver Operator Characteristic (ROC) analysis was carried out, and a score of 5.72 was suggested as the score of SMA that is significant for the clinical outcome with lymph node involvement. RESULTS: Overall, the average SMA was 7.29 (range 0.39-16.84). Further analysis showed correlation of SMA with clinical and pathological tumor characteristics, i.e. SMA was significantly higher in tumors with more advanced stage, higher histological grade, greater amount of desmoplasia, smaller amount of inflammatory infiltrate, lymph node involvement, vascular and perineural invasion and infiltrative tumor growth. CONCLUSION: Our study suggests that it is possible to define the tumor capacity for invasion and metastasis by quantifying the myofibroblasts in the tumor stroma of colorectal carcinomas. Therefore, further investigations are needed to determine targeted therapies to signaling pathways in myofibroblasts.

A paradigm for the control of upright standing in paraplegic patients.

The objective of this paper is to present a new paradigm in control strategy for unsupported paraplegic standing, based on closed-loop control of paraplegics' lower extremities. The main advantage of our approach is taking into account voluntary motions of the upper-part of the body by controlling Center of Mass (CoM) position. The validity of our approach is tested, in computer simulations, using human CoM trajectories estimated from experimental data and by applying perturbations in simulation during quiet standing in order to simulate voluntary upper body movements. From the results presented in this study it can be seen that controller is able to track desired CoM position with sufficient precision and to maintain stability even in the presence of simulated movements of the upper body.

Evaluation of the Beck Depression Inventory in a nonclinical student sample.

Depression is one of the most common psychological disorders in individuals seeking psychiatric treatment, and a frequent psychological disorder among patients who seek primary healthcare. Therefore, it is vitally important to employ reliable and valid diagnostic instruments and norms, both in clinical and research work to investigate this problem. This article is part of a larger study which has been conducted for ten years now with the aim to create a clearer picture about the level of depression which may be expected in the nonclinical population in Serbia, and in that way provide a basis for comparisons when diagnosing the clinical population. The subsidiary aims were to monitor potential changes in level of depressive reactions within the set time and to examine the psychometric properties and factor structure of the Beck Depression Inventory (BDI) scale. The sample consisted of 782 students (40% male, 60% female), mean age = 23.10 years, SD = 1.782. Mean score on the BDI-IA scale was 6.69; SD = 6.412. The study showed no significant relationships between the BDI scores and sociodemographic variables such as age, economic status, and educational profile, but showed significant differences within gender (t (780) = 3.222, p = 0.001). There was also a relatively stable level of depressive reactions in this population over the previous ten years. The Cronbach's coefficient of the BDI scale was alpha = 0.860, with the majority of item-total correlations above 0.37. The three-factor structure represents cognitive aspect, affective component of depression, and somatic problems attached to depression. The cognitive factor prevails in the entire sample, which is in accordance with the Beck theory about dysfunctional attitudes, ie cognitive vulnerability is a psychological predisposition to depression.

First Report of the Occurrence of Cucurbit aphid-borne yellows virus on Oilseed Pumpkin in Serbia.

In July 2008, field-grown oilseed pumpkins (Cucurbita pepo L. 'Olinka') showing severe yellowing and thickening of older leaves were observed in the Kisac locality of Vojvodina Province, Serbia. Symptomatic plants were found only near the borders of the field. Leaf samples collected from 15 symptomatic plants were tested for the presence of four viruses causing the cucurbit yellowing disorder. Total RNAs were extracted from deep frozen plant materials with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and reverse transcription (RT)-PCR was conducted with the OneStep RT-PCR Kit (Qiagen) following the manufacturer's instructions. RNA extracted from healthy C. pepo and molecular-grade water were included as negative controls in each PCR reaction. Species-specific primers (1,2) failed to detect the presence of three viruses causing the cucurbit yellowing disorder, Cucumber vein yellowing virus, Cucumber yellow stunting disorder virus, and Beet pseudo-yellows virus, in symptomatic samples. When two different sets of CABYV-specific primer pairs, CABYVup/CABYVdown (2) and CE9/CE10 (3), for a 484-bp and a 600-bp fragment of the CP gene of Cucurbit aphid-borne yellows virus (CABYV), respectively, were used for amplification, the former amplified fragments of the expected size from all symptomatic samples, whereas the latter successfully amplified a 600-bp fragment from only 7 of 15 samples. The 600-bp amplified product derived from isolate 145-08 was purified (QIAquick PCR Purification Kit, Qiagen), sequenced in both directions, deposited in GenBank (Accession No. HQ202745), and subjected to sequence analysis by MEGA4 software. Sequence comparisons revealed a high nucleotide identity of 99.8% (100 and 99.5% amino acid identities for the CP and the overlapping MP genes, respectively) with Czech CABYV isolates from C. pepo 'Ovifera' (HM771271-73). A neighbor-joining tree obtained on a 545-bp CP fragment of CABYV isolates available in GenBank database revealed that Serbian CABYV isolate 145-08 was clustered with isolates from Spain, Italy, France, and Tunisia in the Mediterranean subgroup denoted previously (4). In a persistent type transmission test, which was carried out using Aphis gossypii Glover, the aphids were allowed to feed on leaves of the collected sample (145-08) for an acquisition access period of 2 days and then 10 aphids were transferred to each of 20 C. pepo 'Olinka' plants for a 5 day inoculation access period. Transmission was successful in 6 of 20 plants as assessed by the development of a mild yellowing symptom 2 weeks after transmission and confirmed by RT-PCR with the CABYVup/CABYVdown primers. To our knowledge, this is the first record of the occurrence of CABYV in Serbia. The discovery of CABYV on oilseed pumpkin should prompt more detailed surveys and subsequent testing of other cucurbits cultivated in Serbia to establish the distribution and incidence of CABYV in Serbia. References: (1) K. Bananej et al. Plant Dis. 90:1113, 2006. (2) I. N. Boubourakas et al. Plant Pathol. 55:276, 2006. (3) M. Juarez et al. Plant Dis. 88:907, 2004. (4) Q. X. Shang et al. Virus Res. 145:341, 2009.

First Report of Plasmopara obducens on Impatiens walleriana in Serbia.

In May 2010, Impatiens walleriana plants with single or double flowers that were showing symptoms resembling those of downy mildew were collected in a greenhouse in the vicinity of Mionica, Kolubara District, Serbia. Diseased plants were severely stunted, with mild inconspicuous mottling and yellowing on the upper surface of the leaves. The lower surface of the affected leaves was completely covered with distinctive thick, white fungal-like growth. Symptomatic leaves wilted very quickly and premature leaf fall was common, leaving plants with only a few of the youngest leaves and no or few and poorly developed flowers. Disease incidence was extremely high, approaching 100%, and wilting and collapse of affected plants was very rapid, resulting in losses of more than 90%. White downy growth developing on leaf undersurfaces consisted of hyaline, thin-walled sporangiophores with monopodial branching and numerous, ovoid and hyaline sporangia. Apical branchlets of sporangiophores were at right angles to the main axis, with no apical thickening. Downy mildew of impatiens can be caused by Plasmopara obducens or the less known Bremiella sphaerosperma. The two can be differentiated on the basis of symptomatology and morphology of sporangiophores (1). The absence of well-defined spots on the infected impatiens leaves and straight sporangiophores indicated that the pathogen was P. obducens, which was further supported by molecular identification. Total DNA was extracted directly from plant tissue with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions, and the 5'-end of the nuclear DNA coding for the large ribosomal subunit (LSU rDNA) was amplified by PCR using primers NL1 and NL4 (3). Each PCR amplification yielded two bands estimated at 800 and 650 bp, respectively. A representative isolate, 28-10, was sequenced and 727 bp of the larger band (GenBank Accession No. HQ246451) were found to be identical with P. obducens isolate (AY587558) from the United Kingdom. The sequence was almost identical with those of three P. obducens isolates deposited in NCBI GenBank: EF196869 and AY035522 differed from it by one base pair and FJ787308 by two base pairs. The sequence (HQ223336) of the smaller band was identical to that of three Impatiens accessions (AY727936, AF479154, and AY056515). Pathogenicity tests included inoculation of young I. walleriana plants by spraying with a sporangial suspension. The inoculated plants were kept in experimental chambers at 20°C and 80 to 90% relative humidity, and downy mildew symptoms were observed after 13 to 15 days. To our knowledge, this is the first report of downy mildew of I. walleriana caused by P. obducens in Serbia. So far the presence of P. obducens was recorded in Bulgaria, the Czech Republic, Denmark, Finland, Lithuania, Romania, Russia, the United Kingdom (2), and recently in Norway (4). Thorough inspection would be needed to determine the distribution and incidence of P. obducens on impatiens in Serbia both indoors and outdoors. Impatiens is one of the most popular ornamentals in Serbia and intensive and increasing production may be seriously endangered by the presence of P. obducens. References: (1) O. Constantinescu. Mycologia 83:473, 1991. (2) C. R. Lane et al. Plant Pathol. 54:243, 2005. (3) W. Maier et al. Can. J. Bot. 81:12, 2003. (4) B. Toppe et al. New Dis. Rep. 20:33, 2010.

First Report of Tomato spotted wilt virus on Gerbera hybrida in Serbia.

In May 2009, approximately 30% of plants within a greenhouse-grown Gerbera hybrida crop in Vranjska Banja (Pcinj District) in Serbia displayed chlorotic oak-leaf patterns followed by necrosis and distortion of leaves. Symptoms on naturally infected gerbera plants and local necrotic spots on Petunia × hybrida mechanically inoculated with infected gerbera sap using chilled 0.05 M phosphate buffer (pH 7) containing 1 mM Na-EDTA, 5 mM Na-DIECA, and 5 mM Na-thioglycolate (4) suggested the presence of a Tospovirus. Symptomatic leaves were tested for the presence of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), and Chrysanthemum stem necrosis virus (CSNV) by commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Loewe Biochemica, Sauerlach, Germany). Commercial positive and negative controls and extract from healthy gerbera tissue were included in each ELISA. All 20 tested plants were negative for INSV and CSNV. TSWV was detected serologically in 18 of 20 gerbera samples. The presence of TSWV in ELISA-positive symptomatic gerbera plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was conducted with the OneStep RT-PCR Kit (Qiagen) using Serbian tobacco TSWV isolate (GQ279731) and RNA extract from healthy gerbera as positive and negative controls, respectively. Two different sets of TSWV-specific primers, L1 TSWVR/L2 TSWVF (2) and M962/M66 (3), for a 276-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene and a 897-bp fragment of the NSm gene, respectively, were used for both amplification and sequencing. RT-PCR analyses of each tested plant detected the presence of amplification fragments of expected size. The amplified products corresponding to part of the RdRp and NSm genes derived from the isolate 158-Gerb were purified (QIAquick PCR Purification Kit, Qiagen) and sequenced in both directions (GenBank Accession Nos. HQ246452 and HQ246453, respectively). Sequence analysis of the partial RdRp gene, conducted using MEGA4 software, revealed 91.1 to 98% nt identity (95.1 to 98.8% amino acid [aa] identities) with corresponding sequences of TSWV L RNA deposited in GenBank. The highest identity was found with an isolate from globe artichoke (AM940436) in Greece, and isolates from tomato (GQ279732), impatiens (GQ132190), and tobacco isolates (GQ279731, FJ189392, and FJ189393) found within Serbia. Analysis of the NSm sequence of isolate 158-Gerb demonstrated nucleotide identities varying between 90.6 and 99.6% (80.9 and 99.6% aa identities) with those of previously reported TSWV isolates. The highest identity was with tobacco isolate GQ373174 from Serbia. Therefore, while gerbera is one of the principal ornamental hosts of TSWV in the EPPO region (1), to our knowledge, this is the first report infecting gerbera in Serbia, which may have a devastating influence on its production. References: (1) Anonymous. OEPP/EPPO Bull. 29:465, 1999. (2) R. A. Mumford et al. J. Virol. Methods 46:303, 1994. (3) W. P. Qiu et al. Virology 244:186, 1998. (4) P. Roggero et al. Plant Dis. 86:950, 2002.

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.

First Report of Iris yellow spot virus on Onion (Allium cepa) in Serbia.

Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae) is established in several European countries (France, Italy, The Netherlands, Poland, Slovenia, Spain, and the UK) and its distribution in the EU region has increased since 2002 (3). In July 2007, symptoms resembling those of IYSV were observed in an onion (Allium cepa) seed crop in the Sirig locality in Serbia. Onion plants exhibited characteristic symptoms of chlorotic or necrotic spindle and diamond-shaped lesions on the leaves and scapes. Symptomatic plants were found throughout the field and disease incidence was estimated at 80%. Leaf and scape samples were tested for the presence of IYSV and two other tospoviruses, Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV), using commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Loewe Biochemica, Sauerlach, Germany). All samples tested negative for TSWV and INSV. IYSV was detected serologically in 26 of 34 onion samples. To determine an experimental host range, samples of IYSV-infected onion plants were homogenized in chilled 0.05 M phosphate buffer pH 7 containing 1 mM Na-EDTA, 5 mM Na-DIECA, and 5 mM Na-thioglycolate (2), and host plants were inoculated with the sap. Mechanical transmission of the virus occurred rarely. All inoculated test plants were assayed by DAS-ELISA and only four species tested positive for IYSV, but not in all replications. Inoculated Chenopodium quinoa developed local chlorotic lesions, Nicotiana tabacum cvs. Samsun and Prilep showed mild mosaic, while infected N. benthamiana were symptomless. For further confirmation of IYSV, conventional reverse transcription (RT)-PCR was performed on extracts made from symptomatic onion leaf material and from the ELISA-positive symptomless leaves of N. benthamiana. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was carried out with the OneStep RT PCR Kit (Qiagen) following the manufacturer's instructions. The primer pair, IYSV56U/IYSV917L, covering the entire nucleocapsid (NC) gene was used for both amplification and sequencing (1). A product of the correct predicted size (896 bp) was obtained from each of the plants assayed, and that derived from isolate 605-SRB was purified (QUIAqick PCR Purification Kit, Qiagen) and sequenced (GenBank Accession No. EU586203). BLAST analyses revealed 86 to 97% sequence identity with the NC gene from all other IYSV. The highest identity (97%) was with leek and onion isolates (GenBank Accession Nos. EF427447 and EF19888) from Spain. To our knowledge, this is the first report of IYSV infection of onion seed crop in Serbia. Thorough inspections and subsequent testing would be needed to establish the distribution and incidence of IYSV in Serbia. References: (1) I. Robène-Soustrade et al. Plant Pathol. 55:288, 2006. (2) P. Roggero et al. Plant Dis. 86:950, 2002. (3) C. Sansford and J. Woodhall. Pest Risk Analysis for Iris Yellow Spot Virus. Online publication. Central Science Laboratory, Sand Hutton, UK, 2007.

Behavioural effects of parafascicular thalamic lesions in an animal model of parkinsonism.

We recently reported that the centromedian-parafascicular thalamic complex (CM-Pf) degenerates in Parkinson's disease and progressive supranuclear palsy. The contribution of such thalamic pathology to disease symptoms has not yet been established. The present study therefore investigated the behavioural impact of lesioning the corresponding thalamic region (termed Pf) on a range of behaviours present in rodents. There were four surgical groups: (1) sham medial forebrain bundle (mfb)+sham Pf, (2) 6-OHDA mfb lesion+sham Pf, (3) sham mfb+NMDA Pf lesion, (4) 6-OHDA+NMDA Pf lesions. Posture, sensory functions and apomorphine-induced rotational asymmetry were assessed before and after each surgery. Other assessments performed including a timed motivational task, grooming behaviours and piloerection. 6-OHDA lesions induced postural (ipsilateral curling and head position biases), sensorimotor (increased latency to respond to tactile stimulation of the contralateral side when eating or grooming) and rotational abnormalities (contralateral circling after apomorphine). The main effects of combined 6-OHDA+Pf lesions were improved performance in a motivational task (decreased latency to retrieve reward) but worsened piloerection, relative to animals with either 6-OHDA or Pf lesions alone. The thalamic zone common to all lesioned animals involved the posterior Pf. Our data suggests that the posterior CM-Pf may be involved in motivational responses and autonomic dysfunction in parkinsonian disorders.

Characteristics of dystonic movements in primary and symptomatic dystonias.

OBJECTIVE: To compare clinical characteristics of the involuntary movements in primary and symptomatic dystonias. PATIENTS AND METHODS: 132 consecutive patients with the diagnosis of primary dystonia and 51 consecutive patients with secondary dystonia caused by well defined structural lesion(s) of the central nervous system, with particular emphasis on the characteristics of involuntary movements. RESULTS: Eight variables with the highest risk contribution to either symptomatic or primary dystonias were identified: dystonic movement in secondary dystonia was much more frequently presented at rest, whereas the presence of dystonic tremor, chronic inflammatory process, or peripheral trauma located in the region that is later affected by dystonia, as well as the use of sensory tricks and development of spontaneous remissions, classified the affected patients more often in the category of those with primary dystonia. CONCLUSION: The study identified several clinical features that may be helpful in differentiating primary from secondary dystonia.