Table 1.
Primer-pairs and best BLAST hits for the selected markers.
Table 2.
List of bacterial strains used in this study.
Figure 1.
Genome map of X. euvesicatoria str. 85-10.
Circles, from the outside in, show: genome coordinates (bp), selected DNA markers (orange), phage related ORFs (black), IS elements (green), tRNAs (red), recombinases (purple), integrases (yellow) and transposases (blue). The GC content, Codon Adaptation Index (CAI) and normalized CAI (CAI/eCAI) values are shown for each marker and for four housekeeping genes.
Figure 2.
The selected primer-pairs were tested for efficiency using eight different Xeu strains. For each assay, three different annealing temperatures were tested: 57°C, 59°C and 61°C.
Figure 3.
Dot blot validation of selected probes.
Nine probes were evaluated with total DNA from a collection of BSX, consisting of 19 Xeu, five Xv, three Xg and two Xp strains. Probability values, obtained with a customized MATLAB algorithm for the automatic data analysis, are detailed in Table 3.
Figure 4.
Neighbor-Joining Tree based on the concatenated sequences of four housekeeping genes of several Xanthomonas.
The sequences of the housekeeping genes atpD, dnaK, efp and gyrB were concatenated and used to infer the MLST profile of X. euvesicatoria and X. vesicatoria strains used in this study, which are highlighted in yellow. The Neighbor-Joining tree was derived from the TN93+G+I model and a bootstrap analysis of 1000 replicates.
Table 3.
Outputted probability values concerning the dot blot validation assays with a collection of BSX strains.
Figure 5.
Detection of BSX in infected plant material using a duplex PCR (markers XV7 and XV11).
Tomato and pepper plants inoculated with Xeu 905, Xv 919, Xg 962 and Xp 4321were processed after one and two weeks to obtain crude bacterial suspensions used as PCR templates. Plants inoculated with Pst DC3000 were used as controls. M – DNA marker (GeneRuler DNA Ladder Mix); Ø-Duplex PCR using distilled water as template; C- healthy tomato and pepper plants.
Figure 6.
Detection of BSX in infected plant material using an inverted dot blot platform.
Crude bacterial suspensions, obtained from tomato and pepper plants leaves after one and two weeks of infection with Xeu 905, were used as templates for PCR enrichment using the markers' primer pairs. PCR products corresponding to each plant were labeled with Digoxigenin and used as probes. Purified DNA from Xeu 905 was used as positive control. Negative controls consisted of tomato plants infected with Pst DC3000 for 2 weeks and uninfected plants. The raw ChemiDoc captures and processed images, using the automatic image analysis algorithm, are shown.