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Detection of Clavibacter michiganensis subsp. michiganensis (Cmm), causing bacterial canker of tomato, was verified using PTA-ELISA and IFAS with PAbs of Neogen Europe Ltd. (UK), and with published and also laboratory-generated PCR primers from the Cmm tomatinase gene. The specificity of this technique was determined with 15 plant-pathogenic and 4 common, saprophytic bacteria. With IFAS, crossreactions were found for Pantoea dispersa, P. agglomerans and Rahnella aquatilis, and with PTA-ELISA for Curtobacterium flaccumfaciens, Pectobacterium atrosepticum and Dickeya sp. Cross-reactions with subspecies other than michiganensis were also found using both methods. Molecular methods were optimized by verification of annealing temperatures and times for both primers. Conditions were finally adjusted to 30 s at 65 degrees C for Dreier's and 10 s at 69 degrees C for our primer set. After this optimization, both primer pairs produced positive reaction only with Cmm. By means of PTA-ELISA and IFAS, Cmm strains were detected at a concentration up to 10(5) CFU/mL and 10(3) CFU/mL, respectively. The PCR test with bacterial cell suspensions reached a sensitivity of 10(3) CFU/mL with our designed primers and 104 CFU/mL with Dreier's primer pair.

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The 16S-23S ribosomal internal transcribed spacer (ITS1) is often used as a subspecies or strain-specific molecular marker for various kinds of bacteria. However, the presence of different copies of ITS1 within a single genome has been reported. Such mosaicism may influence correct typing of many bacteria and therefore knowledge about exact configuration of this region in a particular genome is essential. In order to screen the variability of ITS1 among and within Pseudomonas syringae genomes, an oligonucleotide microarray targeting different configurations of ITS1 was developed. The microarray revealed seven distinct variants in 13 pathovars tested and detected mosaicism within the genomes of P. syringae pv. coronafaciens, pisi, syringae and tabaci. In addition, the findings presented here challenge the using of rRNA analysis for pathovar and strain determination.

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Erwinia amylovora [(BURRILL) WINSLOW et al.] (Ea), the causal agent of fire blight, was detected in plant samples and pure bacterial cultures by means of PCR, IFAS and ELISA. Polyclonal antibodies of Neogen Europe Ltd. were used for IFAS and PTA-ELISA and laboratory-generated primers EaF72 and EaR560 for PCR. Using the BIOLOG system and an immature pear fruit assay, identities of all Ea strains were confirmed as the fire blight bacterium. In assays of pure Ea cultures, PTA-ELISA, and both IFAS and PCR were sensitive to concentrations 10(6)-10(5) and 10(5)-10(4) CFU/mL, respectively. When saprophytic bacteria associated with Ea in plant samples were tested as potentially cross-reacting bacteria, PTA-ELISA and IFAS gave 20 and 14 % cross-reactions, respectively. In plant samples, the presence of Ea was more reliably detected by IFAS (at a dilution of 1 : 1000) than by PTA-ELISA (to dilution 1 : 100). The capacity to detect Ea might be increased using an optimized PCR, but for PCR prepared from infected plant samples it was necessary to use the bacterial DNA isolated with a DNeasy Plant Mini Kit (Qiagen). In this case the PCR was sensitive to a concentration of 10(5) CFU/mL. PCR was much more specific than either immunochemical technique, because no false positives were observed when primers EaF72 and EaR560 were used.

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A total of 20 putative strains of Erwinia amylovora originating from 11 samples of host plants with symptoms of fire blight were analyzed in detail using commercial polyclonal antibodies in immunochemical tests. Fourteen strains reacted negatively in all tests; 6 strains reacted positively with a polyclonal antibody for PTA-ELISA (plate-trapped antigen-enzyme linked immunosorbent assay) at a concentration corresponding to A620 = 0.1, while at A620 readings of 0.01 and 0.001 the results were negative. Five strains reacted positively with a polyclonal antibody for indirect immunofluorescence test at all tested concentrations. Three of those strains were positive in the PCR test with AMSbL and AMSbR primers designed for detection of E. amylovora. In hypersensitivity test in tobacco and in immature pear fruit assay, all putative strains were negative while a known reference strain of E. amylovora gave a typical hypersensitive-reaction response. On a medium with 5% sucrose the reference strain of E. amylovora produced levan while putative strains did not. After modification of the PCR protocol, 3 putative strains reacted as negatives. Optimization of PCR test was achieved by finding the optimum annealing temperature and time for primers. The recommended annealing temperature (49 degrees C) for these primers was increased to 55 degrees C and the annealing time was reduced from 2 min to 30 s. Using the microbial identification system Biolog those 3 strains were identified as Pantoea dispersa (1 strain) and Pantoea agglomerans (2 strains). The strains are supposed to be white variants of the species P. dispersa and P. agglomerans occurring less frequently than the yellow variants. Since there were positive reactions in our immunochemical tests these strains could cause false positives in routine screening of plant samples.

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Microarrays are one of the new emerging methods in plant virology currently being developed by various laboratories. In this study, a new approach is described on the detection of plant viruses using short synthetic single-stranded oligomers (40 nt) instead of PCR products as capture probes. A microchip detecting potato viruses, PVA, PVS, PVM, PVX, PVY and PLRV, in both single and mixed infections was developed and tested. The chip was also designed to distinguish between the main strains of PVY and PVS. Results of initial tests with PVY(NTN) and PVY(O) strains using several different probes for one virus are presented. Possibilities and advantages of the new oligonucleotide-based microarray approach for plant viral diagnosis are discussed.

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DNA microarray assay has become a useful tool for gene expression studies. Less frequent is its application to detection of viruses or diagnostics of virus diseases. Here we show design of a microscope slide-based microarray assay for simultaneous identification of several potato viruses. Different primer pairs were designed or adopted to obtain specific amplicons from six potato viruses: Potato virus A (PVA), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), Potato mop-top virus (PMTV) and Potato leaf-roll virus (PLRV). Purified viral DNA probes were spotted on a microscope slide coated with poly-L-lysine. The same primers were used for preparation of fluorochrome-labeled targets. The latter were denatured and hybridized on the microarray slide (chip). An example of simultaneous assay of two pathogens is given and possibilities of practical application of this type of assay are discussed.

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Testing of 23 isolates of Clavibacter michiganensis subsp. sepedonicus for analysis by rep-PCR (using BOX, ERIC, REP primer sets) was used for the purpose of localization of genetic markers for fluid and/or nonfluid strains. None of the primer sets was successful in detecting genetic differences between the isolates and no polymorphism was generated.

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Repetitive-sequence-based polymerase chain reaction (Rep-PCR) method was used for analysis of genetic variability among bacterial populations from different world locations. Collection of 26 Czech and 13 foreign strains of Clavibacter michiganensis subsp. sepedonicus was amplified using BOX primer targeting to repetitive motif occurring in eubacterial genomes. Genetic fingerprints were visually compared and statistically evaluated by cluster analysis. Genetic similarity was estimated to be approximately 80% among all tested strains. Populations of these bacteria seem to be highly homogeneous; potential influence of geographic origin was not confirmed.

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The coat protein (CP) gene of Prunus necrotic ringspot virus (PNRSV) was cloned into pET 16b vector and expressed in Escherichia coli. CP-enriched fractions were prepared from whole cell lysate by differential centrifugation. The fraction sedimenting at 20,000 x g for 30 mins was used for preparation of a rabbit antiserum to CP. This antiserum had a titer of 1:2048 and reacted in a double-antibody sandwich ELISA (DAS-ELISA).

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Leaves of Fragaria ananassa Duch. cv. Redgauntlet with mottle and mild dwarf symptoms were grafted onto F. vesca indicator clones. The youngest leaves developed specific vein banding pattern located preferentially on secondary veins near the edge of the leaves. Electron microscopy of ultrathin sections and negatively stained purified virus preparations from symptom-bearing strawberry leaves revealed presence of different-sized isometric virions. Particles of about 50 nm and 23 nm in diameter were identified as strawberry vein banding virus (SVBV) and tobacco necrosis virus (TNV) D strain. Based on results of electron microscopy, DNA hybridization, enzyme-linked immunosorbent assay (ELISA), and DNA sequencing we propose that the anomalous "leaf edge vein banding" symptoms are caused by a mixed virus infection with SVBV and other viruses such as TNV.

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Simple and reliable procedure for sample preparation and reverse transcription-polymerase chain reaction (RT-PCR) detection of potato virus A (PVA) is described. PVA-specific primers used in the RT-PCR defined a target sequence of 321 bp and did not produce amplification product(s) with potato virus Y.

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Strawberry vein banding virus (SVBV) was detected by polymerase chain reaction (PCR) and dot-blot hybridisation in samples of cultivated strawberry plants originating from central Slovakia and in samples of wild strawberry plants from south-eastern Serbia in Federal Republic of Yugoslavia (FRY). This is the first finding of SVBV in these countries as well as of SVBV in wild strawberry plants in Europe.

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The complete DNA sequence (7876 nucleotides) of strawberry vein banding virus (SVBV) has been determined. Seven open reading frames are detected that potentially code for proteins of calculated weight 37.8; 18.3; 16.6; 56.0; 81.1; 59.0 and 12.6 kDa, respectively. Their position on the viral genome is the same as that of the corresponding proteins on the cauliflower mosaic virus (CaMV) genome. Phylogenetic analysis based on the amino acid sequence of this protein shows a closer relationship of SVBV with CaMV, figwort mosaic virus and carnation etched ring virus than with other caulimoviruses.

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Strawberry vein banding virus (SVBV) isolates from North America, Czech Republic, Norway, and Germany were collected and their variability was determined by dot blot hybridization and confirmed by sequencing of a 431-nucleotide fragment from the middle part of the coat protein gene. Two different substitutions were found between the American and two Czech SVBV isolates, but the other isolates were identical in the compared region to the American isolate. Digoxigenin-labeled probes were prepared from these isolates and used for hybridization with polymerase chain reaction-amplified fragments of 26 Czech SVBV field isolates. No significant differences in the hybridization signal were found with any combination of samples and probes. These results show that the European isolates probably originate from a common ancestor and may have been introduced to Europe from America with planting or breeding material.

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Strawberry vein banding virus (SVBV) is one of seventeen members of the family Caulimoviridae. Natural infection with the virus is known in Fragaria species only. Infections caused by SVBV are often symptomless (1), but their significance increases in mixed infections with strawberry crinkle or strawberry latent C viruses (2,3). This virus has been originally found on strawberries in USA and firstly described by Frazier (4), but it is probably world-wide distributed by planting or breeding materials. SVBV has been observed on cultivated strawberries in North America, Australia, Brazil, Japan (5) and recently in Europe (6,7). The concentration of SVBV in infected plants is usually very low. Its detection by ELISA is impossible because of lack of specific antibodies. Evidence of the caulimovirus nature of SVBV has been confirmed by its circular dsDNA genome, shape and size of viral particles (8), presence of cytoplasmic inclusion bodies typical for caulimoviruses, and distant serological relationship with cauliflower mosaic virus (CaMV, 9). In this paper we present detection of SVBV by combination of two detection methods--polymerase chain reaction (PCR) and dot blot hybridization with a non-radioactive probe.

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A non-radioactive digoxigenin-labelled cDNA probe was prepared from genomic DNA of American isolate No. 45058 of strawberry vein banding virus (SVBV). Five different air-dried SVBV-containing strawberry leaf samples originating from National Clonal Germplasm Repository, Corvallis, USA, reacted positively in dot blot hybridization with this probe. Six of twelve strawberry samples from the Czech Republic exhibiting symptoms of SVBV-like infection gave positive reaction with this probe. Our results confirm the spread of SVBV in Central Europe and introduce the first reliable screening method for this virus.

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Fifty Erwinia herbicola isolates obtained from host plants were examined in an agglutination reaction with antiserum prepared against E. ananas (E. herbicola) strain CCM 2407 antigen of plant origin and with antiserum prepared against Enterobacter agglomerans strain CNCTC M 269 antigen of human origin. In tests with strain CCM 2407 antiserum, 56% isolates showed a positive reaction, while in tests with strain CNCTC M 269 antiserum only 14% isolates showed a positive reaction. Among E. herbicola isolates which showed a positive reaction with CCM 2407 antiserum 18% showed a positive reaction with the CNCTC M 269 antiserum too. Our results confirmed the serological heterogeneity of E. herbicola population. In spite of the difference in the origin of the two antigens used for the preparation of antisera (plant, human; Japan, Czech Republic) our results indicate that some of our E. herbicola strains and E. agglomerans strain CNCTC M 269 are serologically identical.

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