Isolation and characterization of Agrobacterium strains from grapevines in Bulgarian vineyards and wild grapes, V. vinifera ssp. silvestris

Twenty strains of Agrobacterium spp. were isolated from grapevines derived from 9 different grape-growing regions in Bulgaria and from symptomless wild grapes, Vitis vinifera ssp. silvestris, naturally growing in forests of South-East Bulgaria. The isolated strains were examined by biochemical and pathogenicity tests and PCR analysis of chromosomal and plasmid-located genes. Two of the studied strains resemble non-pathogenic A. tumefaciens, but the others were determined as tumorigenic A. vitis. Only one out of 18 A. vitis strains, isolated from grapevines, was found to harbor vitopine Ti plasmid type. Surprisingly, all 4 A. vitis strains isolated from symptomless V. vinifera ssp. silvestris plants, proved to be tumorigenic. Thus, for the first time we report the isolation of pathogenic A. vitis strains from wild grapes. The strict correlation between the results from biochemical and virulence tests and PCR analysis suggests that the chosen PCR procedures can be used efficiently for direct PCR testing for the presence and tumorigenicity of A. vitis in grapevines.

Genetic diversity of Agrobacterium vitis strains, isolated from grapevines and wild grapes in Bulgaria, assessed by Cleaved Amplified Polymorphic Sequences analysis of 16S-23S rDNA

Nineteen tumorigenic Agrobacterium vitis strains isolated from commercial vineyards and wild grapes at different locations in Bulgaria were studied in relation to the Ti plasmid type and chromosomal background. The PCR analysis showed that all but one of the strains harbor an octopine/cucumopine type of Ti plasmid and one carries a vitopine type. The genetic diversity among the studied strains and 20 more A. vitis strains originating from different geographic regions in Europe, Asia, USA and South Africa was assessed by Cleaved Amplified Polymorphic Sequences (CAPS) analysis of 16S-23S rDNA region. The comparison of the obtained CAPS profiles and performed cluster analysis showed a high level of polymorphism among the studied strains distributed in totally 15 different groups within two main clusters. All Bulgarian strains are located in only three groups within one of the clusters. A high level of correlation between the chromosomal background and type of the carried Ti plasmids was found. The performed CAPS analysis demonstrated that all A. vitis strains isolated from wild grapes (V. vinifera ssp. silvestris) showed CAPS profiles identical with a number of strains isolated from commercial vineyards from different vine-growing regions in Bulgaria. A possible origin of this group of strains from an ancestral A. vitis strain, which initially inhabits wild grapes (V. vinifera ssp. silvestris) and later has been disseminated to cultivated grapevines is proposed

Genotyping Vitis vinifera L. cultivars of Cyprus by microsatellite analysis

Twelve native Vitis vinifera L. cultivars of Cyprus were genotyped at 11 highly polymorphic microsatellite markers. The obtained microsatellite allelic profiles allowed precise identification and discrimination of all tested cultivars. Each cultivar had a unique allelic profile. The low PI value (5.1 x 10-9) demonstrated the high descriptive power of the chosen markers for the investigated set of grapevines. Three cases of synonymy of Cyprus cultivars with cultivars grown in other countries under different names were verified: (1) Cyprus Malaga and Muscat of Alexandria, (2) Cyprus Lefkas and Greek Verdzami, and (3) Cyprus Moscato, Bulgarian Tamyanka and Italian Moscato Bianco. The homonymy of Cyprus Sideritis and Greek Sideritis as well as of Cyprus Mavro and Bulgarian Mavrud was shown not to rely on genetic similarity.

Genotyping of Bulgarian Vitis vinifera L. cultivars by microsatellite analysis

A characterization of the Bulgarian grapevine genepool (Vitis vinifera L. cultivars) was initiated through microsatellite analysis. Seventy four wine and table grapevine varieties from the National List of Cultivars, were analyzed at 9 microsatellite loci: VVS2, ssrVvUCH11, ssrVvUCH 29, ssrVrZAG21, ssrVrZAG47, ssrVrZAG62, ssrVrZAG64, ssrVrZAG79 and ssrVrZAG83. The high genetic diversity (78 %) allowed accurate identification and discrimination of the cultivars. The low PI value (1.201 x 10-8) reflects the high discriminative power of the chosen set of markers for the investigated population. Based on the microsatellite allele data, two pairs of old native varieties, Misket Cherven and Misket Vrachanski; Tamyanka and Tamyanka tvarda, were considered distinct cultivars. The synonymy ofTamyanka, Italian Moscato Bianco and Greek Moschato Kerkyras andPamid and Greek Pamidi was verified, while the putative synonymy of Mavrud and Greek Mavroudi Arachovis was rejected.Further utilization of microsatellite profiling in the management of the Bulgarian grapevine genepool is discussed.

Construction of plant transformation vectors carrying beet necrotic yellow vein virus coat protein gene (i) - transformation vectors

Coat protein gene of beet necrotic yellow vein virus (BNYVV) was isolated from inoculated sugar beet roots and leaves of Chenopodium quinoa and Tetragonia expansa, by RT-PCR and imuno capture RT-PCR. Specific primers were made to complement coat protein gene and untranslated leader sequence, so that two fragments were obtained: long (731 bp), which contained coat protein gene and leader sequence, and short (587 bp), with coat protein gene. Fragments were cloned in two plant transformation vectors: pCAMBIA 3301M and pCAMBIA 1304M, which were modified by removing multicloning site and NcoI restriction site at the 5' end of the reporter genes. Vector pC3301M had bar gene which confers resistance against the herbicide gluphosinate ammonium as selectable marker, and pC1304M had gene for resistance to antibiotic hygromycin. Three constructs were made from each vector: CPL, containing coat protein gene with leader sequence; CPS with gene for coat protein, and CPSas with coat protein gene in antisense orientation. All constructs were transfered to Agrobacterium tumefaciens strain LBA4404

Genetic diversity in native Bulgarian grapevine germplasm (Vitis vinifera L.) based on nuclear and chloroplast microsatellite polymorphisms

Fifty one wild specimens collected in different areas in Bulgaria and nineteen native Bulgarian grapevine cultivars were genotyped with 7 nuclear and 5 chloroplast SSR markers. Based on the microsatellite allelic profile six wild samples, collected from the Danube Riverbank, were considered non vinifera genotypes. The genetic diversity for nuclear loci observed in the cultivated grapevines was comparable to that found in other cultivated collections. However, lower genetic diversity was observed in the set of wild samples. The dendrogram based on nuclear SSRs separated most of the cultivated grapevines from the wild samples. Four chlorotypes corresponding to previously determined chlorotypes A, B, C and D, were identified in the analyzed samples that occurred with different frequencies in groups of wild and cultivated plants. The most frequent chlorotype among wild samples was A, while it was C in the cultivated samples. The differentiation of Bulgarian grape chlorotypes in the context of differentiation of chlorotypes in Eurasian grape flora is discussed.

Increased power generation in supercapacitive microbial fuel cell stack using Fe-N-C cathode catalyst

The anode and cathode electrodes of a microbial fuel cell (MFC) stack, composed of 28 single MFCs, were used as the negative and positive electrodes, respectively of an internal self-charged supercapacitor. Particularly, carbon veil was used as the negative electrode and activated carbon with a Fe-based catalyst as the positive electrode. The red-ox reactions on the anode and cathode, self-charged these electrodes creating an internal electrochemical double layer capacitor. Galvanostatic discharges were performed at different current and time pulses. Supercapacitive-MFC (SC-MFC) was also tested at four different solution conductivities. SC-MFC had an equivalent series resistance (ESR) decreasing from 6.00 Ω to 3.42 Ω in four solutions with conductivity between 2.5 mScm−1 and 40 mScm−1. The ohmic resistance of the positive electrode corresponded to 75–80% of the overall ESR. The highest performance was achieved with a solution conductivity of 40 mS cm−1 and this was due to the positive electrode potential enhancement for the utilization of Fe-based catalysts. Maximum power was 36.9mW (36.9Wm−3) that decreased with increasing pulse time. SC-MFC was subjected to 4520 cycles (8 days) with a pulse time of 5 s (ipulse 55 mA) and a self-recharging time of 150 s showing robust reproducibility

Impact of catalyst layer morphology on the operation of high temperature PEM fuel cells

Electrochemical impedance spectroscopy (EIS) is a well-established method to analyze a polymer electrolyte membrane fuel cell (PEMFC). However, without further data processing, the impedance spectrum yields only qualitative insight into the mechanism and individual contribution of transport, kinetics, and ohmic losses to the overall fuel cell limitations. The distribution of relaxation times (DRT) method allows quantifying each of these polarization losses and evaluates their contribution to a given electrocatalyst\u27s depreciated performances. We coupled this method with a detailed morphology study to investigate the impact of the 3D-structure on the processes occurring inside a high-temperature polymer electrolyte membrane fuel cell (HT-PEMFC). We tested a platinum catalyst (Pt/C), a platinum-cobalt alloy catalyst (Pt$_{3}$Co/C), and a platinum group metal-free iron-nitrogen-carbon (Fe–N–C) catalyst. We found that the hampered mass transport in the latter is mainly responsible for its low performance in the MEA (along with its decreased intrinsic performances for the ORR reaction). The better performance of the alloy catalyst can be explained by both improved mass transport and a lower ORR resistance. Furthermore, single-cell tests show that the catalyst layer morphology influences the distribution of phosphoric acid during conditioning

Development of Grid e-Infrastructure in South-Eastern Europe

Over the period of 6 years and three phases, the SEE-GRID programme has established a strong regional human network in the area of distributed scientific computing and has set up a powerful regional Grid infrastructure. It attracted a number of user communities and applications from diverse fields from countries throughout the South-Eastern Europe. From the infrastructure point view, the first project phase has established a pilot Grid infrastructure with more than 20 resource centers in 11 countries. During the subsequent two phases of the project, the infrastructure has grown to currently 55 resource centers with more than 6600 CPUs and 750 TBs of disk storage, distributed in 16 participating countries. Inclusion of new resource centers to the existing infrastructure, as well as a support to new user communities, has demanded setup of regionally distributed core services, development of new monitoring and operational tools, and close collaboration of all partner institution in managing such a complex infrastructure. In this paper we give an overview of the development and current status of SEE-GRID regional infrastructure and describe its transition to the NGI-based Grid model in EGI, with the strong SEE regional collaboration.Comment: 22 pages, 12 figures, 4 table

Deregulated splicing is a major mechanism of RNA-induced toxicity in Huntington's disease

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin (HTT) gene, translating into an elongated polyglutamine stretch. In addition to the neurotoxic mutant HTT protein, the mutant CAG repeat RNA can exert toxic functions by trapping RNA-binding proteins. While few examples of proteins that aberrantly bind to mutant HTT RNA and execute abnormal function in conjunction with the CAG repeat RNA have been described, an unbiased approach to identify the interactome of mutant HTT RNA is missing. Here, we describe the analysis of proteins that preferentially bind mutant HTT RNA using a mass spectrometry approach. We show that (I) the majority of proteins captured by mutant HTT RNA belong to the spliceosome pathway, (II) expression of mutant CAG repeat RNA induces mis-splicing in a HD cell model, (III) overexpression of one of the splice factors trapped by mutant HTT ameliorates the HD phenotype in a fly model and (VI) deregulated splicing occurs in human HD brain. Our data suggest that deregulated splicing is a prominent mechanism of RNA-induced toxicity in HD