Characterization and PCR-based Typing of Xanthomonas campestris pv. vesicatoria from Peppers and Tomatoes in Serbia

During the last two decades bacterial strains associated with necrotic leaf spots of pepper and tomato fruit spots were collected in Serbia. Twenty-eight strains isolated from pepper and six from tomato were characterized. A study of their physiological and pathological characteristics, and fatty acid composition analysis revealed that all of the strains belong to Xanthomonas campestris pv. vesicatoria. Being non-amylolytic and non-pectolytic, pathogenic on pepper but not on tomato, containing lower amounts of fatty acid 15 : 0 ante–iso, the pepper strains were designated as members of the A group of X. campestris pv. vesicatoria. However, the tomato strains hydrolyzed starch and pectate, caused compatible reactions on tomato but not on pepper, had higher percent of 15 : 0 ante–iso fatty acid, and were classified into B phenotypic group and identified as X. vesicatoria. PCR primers were developed which amplified conserved DNA regions related to the hrp genes of different strains of X. campestris pv. vesicatoria associated with pepper and tomato. Restriction analysis of the PCR product resulted in different patterns and enabled grouping of the strains into four groups. When xanthomonads isolated from pepper and tomato in Serbia were analyzed, they clustered into two groups corresponding to the grouping based on their physiological and pathological characteristics. According to the reaction of pepper and tomato differential varieties, the strains from pepper belong to races P7 and P8 and tomato strains belong to the race T2. All strains were sensitive to copper and streptomycin. Advantages and disadvantages of various bacterial spot management practices are discussed.     

A Data Generator for Evaluating Spatial Issues in Precision Agriculture

With the rapid rise in site-specific data collection, many research efforts have been directed towards finding optimal sampling and analysis procedures. However, the absence of widely available high quality precision agriculture data sets makes it difficult to compare results from separate experiments and to assess the optimality and applicability of procedures. To provide a tool for spatial data experimentation, we have developed a spatial data generator that allows users to produce data layers with given spatial properties and a response variable (e.g. crop yield) dependent upon user specified functions. Differences in response functions within fields can be simulated by assigning different models to regions in coordinate-(x and y) or feature space (multidimensional space of attributes that may have an influence on response). Noise, either unexplained variance or sensor error, can be added to all spatial layers. Sampling and interpolation error is modeled by sampling a continuous data layer and interpolating values at unsampled locations. The program has been successfully tested for up to 15000 grid points, 10 features and 5 models. As an illustration of the potential uses of generated data, the effect of sampling density and kriging interpolation on neural network prediction of crop yield was assessed. Yield prediction accuracy was highly related (correlation coefficient 0.98) to the accuracy of the interpolated layers indicating that unless data are sampled at very high densities relative to their geostatistical properties, one should not attempt to build highly accurate regression functions using interpolated data. By allowing users to generate large amounts of data with controlled complexity and features, the spatial data generator should facilitate the development of improved sampling and analysis procedures for spatial data.     

Identification and characterization of Agrobacterium spp. isolated from apricot in Serbia

Crown gall, caused by tumorigenic strains of Agrobacterium spp., is considered one of the most important diseases in stone fruit nurseries throughout the world. Since the crown gall disease has not been studied extensively in Serbia for more than 30 years, the objective of this study was to isolate, identify and characterize the bacterium associated with crown gall symptoms on one-year-old apricot trees. Samples were collected from a nursery in central Serbia and subjected to laboratory analysis. Bacteria were isolated from tumour tissue on yeast mannitol agar (YMA) and six gram-negative isolates were selected for further study. PCR assay using primers specific for virD2, virC, ipt and tms2 pathogenicity-associated plasmid genes revealed that isolates harbour Ti plasmids. All studied strains carrying Ti plasmids were classified as nopaline-type based on further molecular analysis. Using a multiplex PCR assay, targeting 23S rRNA gene sequences, and physiological and biochemical tests, five strains were assigned as A. rhizogenes/biovar 2 and the remaining one as A. tumefaciens/biovar 1. Identity of the strains was confirmed by sequence analysis of the 16S rRNA gene. In pathogenicity assay, all six strains caused tumour formation on inoculated carrot root discs, young tomato and sunflower plants.     

Exploring diversity of Erwinia amylovora population in Serbia by conventional and automated techniques and detection of new PFGE patterns

Forty Erwinia amylovora strains originating from different host plants and locations in Serbia and one strain from Montenegro were characterized by conventional, automated and molecular techniques. All strains were Gram-negative, nonfluorescent, facultative anaerobes, oxidase negative, levan positive, produced necrotic lesions followed by bacterial exudate on artificially inoculated immature pear fruits and caused HR on tobacco. Based on carbon source utilization, all strains tested with the Biolog system were identified as E. amylovora. Based on fatty acid profiles all tested strains clustered into three groups in which strains from north Serbia differed from strains isolated in central and south parts of the country. Restriction analysis of genomic DNA using XbaI and PFGE resulted in six different patterns differentiating the strains into six groups. Most of the investigated strains clustered in one group having the pattern type similar to Pt2 group described earlier as dominant in East Europe and the Mediterranean region. Two strains showed PFGE pattern similar to the previously described Pt3 pattern and one strain had pattern similar to Pt6. Based on size and number of the bands, new restriction patterns, assigned as Pt7, Pt8 and Pt9 were observed. PFGE results showed that the E. amylovora population in Serbia is not homogenous and was possibly introduced from different directions. This is the first characterization of E. amylovora collection of strains from Serbia using fatty acid analysis and PFGE.     

Characterization and phylogenetic diversity of <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> from Serbia based on sequence analysis of 16S-23S rRNA internal transcribed spacer (ITS) region

Serious outbreaks of grapevine crown gall disease were observed in major Serbian viticultural regions during the last five years. Tumorigenic Agrobacterium vitis was identified as a causal agent by using conventional bacteriological and molecular tests. The 36 studied strains of A. vitis showed homogeneous biochemical and physiological characteristics, but were heterogeneous in their pathogenic properties, especially on tomato and sunflower. Furthermore, genetic differences related to chromosomal and plasmid DNA were observed. The Ti plasmid of 35 strains was classified as the octopine/cucumopine (O/C) type, whereas one was classified as the vitopine (V) type. The O/C strains were further divided into O/C-1 and O/C-2 groups based on PCR analysis. Moreover, the sequence analysis of the 16S-23S rRNA ITS region provided robust and precise delineation of studied strains. Although a high level of genetic diversity in A. vitis strains from Serbia was revealed by using this approach, their genotypic relatedness with the strains from other countries suggested their common origin. Also, association between the chromosomal and plasmid DNA was determined for some phylogenetic groups and clusters.